Microbial Biodiversity in Tasmanian Caves

MICROBIAL BIODIVERSITY

IN

TASMANIAN CAVES

Big Stalagmite, En trance Cave, Tasmania. Photograph taken by Jodie van de Kamp.

Jodie Lee van de Kamp, B.Sc. (Hons)

Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The University of Tasmania Hobart, August, 2004 Declaration I declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in the text of this thesis.

Jodie Lee van de Kamp

25th August 2004

2 Authority of Access

This thesis may be made available for loan and limited copying in accordance with the Copyright Act, 1968.

Jodie Lee van de Kamp

25th August 2004

3 ABSTRACT

Caves represent one of few remaining isolated planetary habitats, in terms of human impact and characterisation of microbial biodiversity. Caves are unique environments characterised by little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate providing ecological niches for highly specialised organisms. Caves are not uniform environments in terms of geological and geochemical characteristics, as they can vary from one to the other, eg. rock type, method of formation, length, depth, number of openings to the surface, presence or absence of active streamways, degree of impact by human visitation etc. Furthermore, on a smaller scale, various microhabitats, with vast differences in community structure can exist within caves. Culture studies point to the dominance of actinomycetes in caves and reveals great taxonomic diversity within actinomycetes isolated. However it is widely accepted that only - 1 % of microbes are cultured in the laboratory. Culture-independent methods are being increasingly used to describe the composition of microbial communities and reveal significantly broader diversity than culture-based studies. Nevertheless, to date our knowledge of bacterial communities in caves is largely due to culture studies. Based on the literature available, this study was initially aimed at examining culturable vs. non-culturable diversity of actinomycetes in Entrance and Loons Caves and to gain an increased understanding of the composition of cave microbial communities employing classical isolation and advanced molecular detection methods. As the study progressed the focus evolved as it became apparent that actinomycetes dominated only very specific habitats, the dry sediment in Entrance Cave, and represented only a minor fraction of the microbial biodiversity of most other microhabitats studied. Entrance Cave dry sediments and inactive (dry) speleothems produced a higher number of actinomycete isolates compared to saturated sediments and wet formations from Entrance and Loons Caves. This was reinforced by the actinomycetes being the second most abundant group (26.8%) detected in clone analysis of the dry Entrance sediment and low abundances (4-16%) detected in saturated sediments from both Entrance and Loons Caves. Sediment phylotypes and isolates identified in this study closely resemble species associated with oligotrophic, chemolithotrophic and heterotrophic lifestyles indicating that these communities survive by utilising a combination of metabolic pathways. Bacteria involved in the nitrogen and sulfur cycles were important members of all sediment communities along with hydrogen-oxidising bacteria. Pair-wise comparisons of sediment communities demonstrated that they were more similar to each other within individual cave systems, Entrance and Loons, rather than between microhabitat types (dry vs. wet sediment) though saturated sediment from Entrance Cave did show a higher degree of similarity in community composition to Loons Cave samples than the dry sediment from Entrance Cave. Saturated sediments were dominated by oligotrophs able to fix atmospheric gases, methanotrophs and had a high proportion of rare phylotypes most likely representing new

4 lineages related to microbes detected in anaerobic, anoxic environments, but low abundances of heterotrophic microbes. Geornicrobiological activities are no longer underestimated since studies have shown that bacterial metabolism may lead to mineral precipitation or dissolution. Questions remain as to the identity of these microbes and whether they are actively involved in speleothem formation, or simply buried during mineral precipitation. Results demonstrated a marked difference between sediment communities and those associated with calcite speleothem and calcite mat samples. Results of ESEM and XRD analysis demonstrated that calcite speleothem samples ME3 and MXl are true calcite moonrnilk (mondmilch). Phylogenetic analyses and isolation results demonstrated the unique composition of the microbial communities associated with moonrnilk deposits, predominantly composed of nitrogen-fixing ~-Proteobacteria and psychrotrophic heterotrophic CFBs and to a lesser extent, heterotrophic actinomycetes. Despite XRD and ESEM analysis showing similar calcite composition and crystal morphology, phylogenetic results indicated that sample ME2 represented a very different rnicrohabitat to moonmilk samples, dominated by oligotrophic a.-Proteobacteria and heterotrophic actinomycetes composing 84.2% of the total diversity. Phylogenetic analyses and biodiversity indices reveal the striking similarities between moonmilk samples from both Entrance and Exit Caves and the uniqueness of the calcite mat in Entrance Cave. The one similarity in composition between all three calcite communities was the presence of members of the Pseudonocardineae in particular of the genus Saccharothrix, in all calcite samples. 165 rRNA gene sequencing of cave isolates detected high levels of diversity and novelty, particularly of moonrnilk isolates. A total of two putatively novel genera (within the CFBs and Actinobacteria) and 18 putatively novel species (of genera: Paracoccus, Actinoplanes I Couchioplanes, Micromonospora, Amycolatopsis, Saccharothrix, Bacillus, Paenibacillus, Methylobacterium, Porphyrobacter, Sphingomonas, Alcaligenes, Stenotrophomonas, Xanthomonas) were identified. This study represents the first reported culture-independent analysis of moonrnilk microbial communities globally and of cave sediment communities in the Southern Hemisphere. Information gained from this study and the discovery of actively growing microbial communities appearing to precipitate CaC03 provides focus for important future studies and represents a unique opportunity to examine the nature and extent of complex microbe-mineral interactions in the formation of speleothems and implications for cave management. The biodiversity described acts as a baseline for assessing environmental impacts and to identify factors influencing microbial biodiversity.

5 ACKNOWLEDGEMENTS I would like to sincerely thank the following people:

The University of Tasmania, Australian Biological Resources Study and Tasmanian Institute of Agricultural Research for funding that not only made this project possible but also allowed the work to be presented at several conferences, both nationally and internationally. National Parks and Wildlife Service, Tasmania, for in-kind support of the project including permits, data and advice.

Supervisors, Dr. David Nichols and Dr. Kevin Sanderson, for managing to capture my interest in the project, open doors for me and remain focused to the end.

Tom McMeekin, Tom Ross, Mark Brown, Adam Smolenski, Sharee McCammon, David Steele, Ralph Bottril, Susan Turner, Olivier Brassiant, Jill Rowling, Bill Cohen, Brendon Bateman and particularly John Bowman and Diana Northup, for technical expertise, excellent advice and helping me find direction when needed.

The School of Agricultural Science, particularly the fantastic Microbiology Group, for providing endless opportunities, support and so many fond and entertaining memories. Including, but not solely, Kathleen Shaw, Guy Abel, Andrew Bisset, Matthew Smith, Shane Powell, Liv McQuestin, Laurie Parkinson, Andy Measham, Jane Weatherly, Heather Haines and Jimmy Twin. Special thanks to Lyndal Mellefont, Kristen Stirling and Craig Shadbolt.

On a personal note, I am truly amazed at the overwhelming support from everyone in my life, I value that friendship more than you'll ever realise. There are so many people to thank, but special notes to, Kriss and Sarah Lawler, Mark van den Berg, Mark Jones, Nat Doran, Hill-Streeters Andy Wilson, Lee-Roy Evans and Kath Fearnley-Sander, Bee Hart, the Marauders, especially my girls and Sonya Enkleman, and for keeping me sane all these years, Tracey Brewer and Miss Holly Taylor.

My wonderful extended family for so much love, support and unquestioning faith that I will succeed. My parents, Lorraine and Peter van de Kamp, my siblings, Jas, Brad, Laura and Steven, and their partners, Megs, Bridg and Justie, who never quite understood why I stayed at 'school' for so long, but have always been there for me.

Finally, and certainly not least of all, Brendon, who always does what he can to help, has put up with me over these last few months without complaining (much©) and most of all is so full of support for the next stage of the journey. Thank you.

6 TABLE OF CONTENTS

MICROBIAL BIODIVERSITY IN TASMANIAN CAVES ...... 1

SECTION 1: ...... 9

LITERATURE REVIEW - MICROBIAL ECOLOGY OF CAVES ...... 9

1.1 MICROBIAL ECOLOGY ...... 9 1.1.1 OBJECTIVES OF MICROBIAL ECOLOGY ...... 9 1.1.2 METHODS IN MICROBIAL ECOLOGY AND TAXONOMY ...... 10 1.1.2.1 BIODIVERSITY ...... 10 1.1.2.2 COMMUNITYFlNGERPRINTING ...... 12 1.1.2.3 ECOLOGICALF'UNCTION ...... 13 1.1.3 LIMITATIONS OF METHODS ...... 16 1.2 CAVES ...... 19 1.2.l SPELEOGENESIS: CAVEFORMATION ...... 19 1.2.2 SPELEOTHEMS: CAVE DECORATION ...... 20 1.2.3 CAVE ENVIRONMENT ...... 21 1.2.4 SPELEOLOGY: CAVE STUDY ...... 22 1.3 MICROBIAL BIODIVERSITY AND ECOLOGY OF CAVES ••••••••••••••••••••••••••••••••••••••••••••••••••• 24 1.3.1 CHEMOLITHOAUTOTROPHIC SYSTEMS ...... 25 1.3.1.1 SULFUR-BASED SYSTEMS ...... 25 1.3.1.2 IRON, MANGANESE, NITRITE, AND OTHER SYSTEMS ...... 27 1.3.2 HETEROTROPHIC SYSTEMS ...... 29 1.3.3 ACTINOMYCETES IN CAVES ...... 31 1.3.3.l ACTINOBACTERIA ...... 36 1.3.3.2 ACTINOMYCETES ...... 36 1.3.3.3 ACTINOMYCETE TAXONOMY ...... 37 1.3.3.4 ACTINOMYCETE ECOLOGY ...... 38 1.4 GEOMICROBIOLOGY•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 40 1.4. l GEOMICROBIOLOGY IN CAVES ...... 40 1.4.2 MICROBIALLY MEDIATED CAC03 PRECIPITATION ...... 43 1.4.3 MOONMILK ...... 46 1.5 SIGNIFICANCE ...... 52 1.5.1 BIODIVERSITY AND CONSERVATION VALUE ...... 52 1.5.2 BIOPROSPECTING ...... 53 1.5.3 BIOREMEDIATION ...... 54 1.5.4 BIODETERIORATION & BIOMINERALISATION PROCESSES ...... 55 1.5.4. l PALAEOLITHIC FRESCOES AND ROCK ART IN HYPOGEAN ENVIRONMENTS ...... 55 ,_ 1.5.4.2 MONUMENTS ...... 56 1.5.5 MANAGEMENT ISSUES ...... 57 ? 1.6 CONCLUSION •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 59

7 SECTION 2: ...... 61

MICROBIAL BIODIVERSITY IN TASMANIAN CAVES ...... 61

CHAPTER 1: INTRODUCTION ...... 61 CHAPTER 2: MATERIALS AND METHODS •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 65 2.1 SITE DESCRIPTION AND SAMPLE COLLECTION ...... 65 2.1.1 ENTRANCE-EXIT CAVE SYSTEM ······················································································ 65 2.1.2 LOONS CAVE ...... 65 2.1.3 SAMPLE COLLECTION ...... 66 2.2 MICROSCOPY ANDMINERALOGY ...... 68 2.2.1 ESEM AND X-RAY ELEMENTAL MICROANALYSIS ...... •...... •...... ~ ... 68 2.2.2 X-RAY DIFFRACTION ANALYSIS ...... 68 2.3 ISOLATION AND IDENTIFICATION OF MICROBES ...... 69 2.3. l ISOLATION AND CULTURING OF MICROBES ...... 69 2.3.2 16S RRNA GENE SEQUENCING AND PHYLOGENETIC ANALYSIS OF ISOLATES ...... 70 2.3.2. l EXTRACTION OF NUCLEIC ACIDS AND PURIFICATION ....•.....•...... 70 2.3.2.2 l 6S RRNA GENE PCR AMPLIFICATION AND PURIFICATION ...... 72 2.3.2.3 16S RRNA GENE SEQUENCING······················································································ 73 2.3.2.4 PHYLOGENETIC ANALYSIS ...... 74 2.4 MOLECULAR ANALYSIS OF SEDIMENTS AND MOONMILK ...... 75 2.4.1 EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS FROM ENVIRONMENTAL SAMPLES 75 2.4.2 DGGE...... 77 2.4.3 CLONE LIBRARY ANALYSIS ····························································································· 79 2.4.4 PHYLOGENETIC AND BIODIVERSITY ANALYSIS ...... 81 CHAPTER 3: RESULTS AND DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 83 3.1 MICROSCOPY AND MINERALOGY ...... 83 3.2 METHOD DEVELOPMENT FOR CALCITE MOONMILK SAMPLES ...... 86 3.3 PHYLOGENETIC DIVERSITY OVERVIEW ...... , ...... 89 3.4 ISOLATION OF NOVEL CAVE MICROBES ...... 130 3.5 DIFFERENCES INMICROHABITATCOMMUNITY STRUCTURE ...... •...... 132 3.6 CULTURABLE VS. NON-CULTURABLE DIVERSITY ...... 138 3.7 METABOLIC/ECOLOGICAL COMPARISONS ...... 143 3.8 COMPARISON WITH OTHER CAVE ENVIRONMENTS ...... 148 CHAPTER 4: CONCLUDING REMARKS ..•.•...••...... ••...... •••...... •...... •••...... •••. 155

REFERENCES ...... 159

APPENDICES ...... 187

APPENDIX 1: MEDIA PREPARATION AND RECIPES ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 187 MEDIA PREPARATION ...... 187 CULTURE MEDIA ...... 187 APPENDIX 2: CRYOPRESERVATION PROTOCOL•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 189

8 1.1 Microbial Ecology

SECTIONl:

LITERATURE REVIEW -MICROBIAL ECOLOGY OF CAVES

1.1 Microbial Ecology

1.1.1 Objectives of Microbial Ecology

Microbial ecology can be defined as investigating the impact of biodiversity on the

structure and function of microbial communities and the ecosystem as a whole. According to

Siering (1998) the questions directing much of the research in microbial ecology are theoretically

quite simple: i) what are the numbers and identities of microorganisms in a given sample, ii)

what are their activities and their role in ecosystem maintenance, iii) what genes are present to

encode the activities of interest, iv) are the genes being expressed (i.e., transcribed), and are

those transcripts translated and processed into active proteins, and v) what controls the rate of

transcription and translation for environmentally significant genes, and can we measure these rates in situ? Much of the recent advances in the field of microbial ecology focuses on addressing

the first question, microbial biodiversity, or, determining the identity of the organisms present

in a given community.

Microbial biodiversity is greater than the diversity of any other group of organisms.

Higher life forms rely on bacterial processes for their survival. Microorganisms are responsible

for diverse metabolic functions that affect soil, plant and animal health, for example, nutrient

cycling, organic matter formation and decomposition, soil structure formation, and plant growth

promotion. Microbial biodiversity has received particular attention in areas where industrial

applications are evident, such as for marine, medical, and food biotechnology, and where

microbial activity has important implications for Earth's climate and for the bioremediation of

polluted sites (Morris et al. 2002).

Different habitats may be characterised by a particular food source, substrate type,

micro-climate, or a combination of these. Some organisms are entirely restricted to a certain

9 1.1 Microbial Ecology habitat whilst others, referred to as cosmopolitan species, range widely across a variety of habitats. Each of these environments/microhabitats has its own characteristics which preclude

generalisations about the conditions of life in one being carried over to the others in most

instances, and which select for bacteria adapted to their own micro-climate.

Enhancing knowledge of bacterial biodiversity and ecological function provides baseline

information for conservation and sustainable development.

1.1.2 Methods in Microbial Ecology and Taxonomy

The study of microbial processes in an ecosystem is a multifaceted affair requiring

attack from many angles and utilising a wide variety of techniques (Brown, 2000). Studies of

biodiversity, characterising the composition of microbial communities in a given environment,

can largely be a descriptive endeavour, but a necessary first step in determining the nature of

biodiversity and its impact on ecological processes.

1.1.2.1 Biodiversity

Traditionally, microbial populations have been described in terms of isolating pure

cultures and investigating a wide range of phenotypic traits, many of which are related to the

practical interest in the habitat studied (eg. phenotypic characteristics of psychrophiles in

Antarctica; Nichols et al. 1993, 1999). Biodiversity studies focusing on phylogenetic or taxonomic

comparisons of microorganims reflect the historical tribulations surrounding the complexities of

defining a bacterial species and the relatedness among individuals of different genotypes

(Morris et al. 2002). Phylogenetic classification of bacteria is based on ancestral relationships

(Woese, 1987). Surprisingly the term 'phylogeny' is rarely defined precisely (Young, 2001). A

central outcome of phylogenetic classification is that taxa be monophyletic, ie. members of a

taxon under consideration share the same common ancestor. A further requirement is that taxa

sharing more recent common ancestry are considered to be more closely related to one another than they are to other taxa (Lincoln et al. 1998 in Young, 2001).

10 1.1 Microbial Ecology A study of microbial biodiversity publications by Morris & co-workers (2002), found that over the last 25 years, DNA-based characterisation techniques, in particular those based on targeted DNA sequences, have had the dominant role in studies of microbial relationships or in the search for new taxa, relative to other morphological or biochemical techniques. By the early

1980s, several studies had shown that ribosomal RNA (rRNA) held promise for phylogenetic reconstruction (Fox et al. 1980) and by the end of the decade, analysis of universally conserved nucleic acid sequences (particularly those of the small subunit rRNA gene) had become a powerful tool for microbial taxonomy, allowing identification of specific taxa on the basis of only a single gene sequence (Woese et al. 1990). In the 1990's, this approach had become the principal method of establishing phylogenetic relationships among the prokaryotes; today, it is more likely that a 165 rRNA gene sequence will be the first piece of data collected for unknown organisms, rather than a Gram stain (Lilburn & Garrity, 2004). Though rRNA methods are now commonplace it is worthwhile to quickly review the basis for this.

There are several reasons to focus on rRNAs to characterise microbial diversity and infer phylogenetic relationships. Olsen et al. (1986) summarised these as follows: i) rRNAs, as key elements of the protein-synthesising machinery, are present, and functionally and evolutionarily homologous, in all organisms, ii) rRNAs are ancient molecules, and conservation of function dictates conservation in overall structure thus, homologous rRNAs are readily identifiable by their size, iii) nucleotide sequences are also conserved allowing comparisons between different organisms and also providing convenient hybridisation targets for cloning and primer directed sequencing techniques, iv) rRNAs constitute a significant component of the cellular mass in actively growing cells (-104 ribosomes per actively growing E. coli cell; Siering, 1998) and are readily detected, isolated and sequenced from all types of organisms, v) rRNAs provide sufficient sequence information to permit statistically significant comparisons, vi) rRNA genes lack artefacts of lateral transfer between contemporaneous organisms. Thus, relationships between rRNAs reflect evolutionary relationships of the organisms. Conservation of function dictates a conservation of structure such that most of the rRNA molecule is conserved among the most divergent or organisms. Although different portions of the molecule evolve at different

11 1.1 Microbial Ecology rates resulting in hypervariable domains as well as highly conserved domains. Their resistance to evolutionary change allows the entire phylogenetic span of ancient and modern prokaryotes to be analysed simultaneously. However it has been shown that the resolution power of rRNA sequences is limited when closely related organisms that diverged at almost the same time are being examined (Woese, 1987; Fox et al. 1992).

It has been estimated that less than 1% of the total bacterial population in a given environment have been successfully isolated (Amann et al. 1995). The advent of culture­ independent molecular methods, especially rRNA-based techniques, led to an explosion of microbial biodiversity papers starting in the late 1980s. Much of what is known is based on distinguishing different organisms as represented by their extracted and polymerase chain reaction (PCR) amplified nucleic acids without actually culturing them or having any direct knowledge of their morphology, physiology or ecology (Kemp & Aller, 2004).

PCR amplification of nucleic acids extracted from environmental samples (eg. soil, water, ice) is at present the most powerful cultivation-independent technique. PCR facilitates the sensitive and fast detection of low amounts of specific gene fragments. This is of particular importance to this study as subsurface environments are, in general, characterised by low biomass which releases low amounts of nucleic acids upon extraction (Chandler et al. 1998).

Microbial diversity and identity can be estimated by cloning, sequencing and phylogenetic analysis of 16S rRNA amplified genes. Clone analysis, more often than not, results in sequences corresponding to previously uncharacterised and often unexpected lineages. An explosion of culture-independent studies of diversity in a wide range of microbial habitats in the past 15 years has resulted in a large database of more than 62 OOO 16S rRNA gene sequences providing a high resolution framework for phylogenetic analysis.

1.1.2.2 Community Fingerprinting

Community fingerprints, may be of use when trying to snapshot the diversity of a population or to follow changes in microbial communities that result from natural community succession, or environmental or anthropogenic perturbation. With specialised computer

12 1.1 Microbial Ecology software fingerprints can be databased and subjected to multivariate statistical analyses (eg.

Roling et al. 2000; Dunbar et al. 2001). Computer assisted analysis allows the comparison of different profiles with each other and the establishment of relationships between fingerprints and environmental conditions (Roling & van Verseveld, 2002). Thus community fingerprinting is more efficient (eg. cost, time) than more detailed clone library analysis when attempting high throughput or comparisons of several communities. Several 16S rRNA gene-based techniques have been used to fingerprint microbial communities, examples of which include, denaturing or temperature gradient gel electrophoresis (DGGE/TGGE), terminal restriction fragment length polymorphism (T-RFLP), and fluorescent in situ hybridisation (FISH).

Although relatively new, DGGE/TGGE is an increasingly popular molecular tool to analyse general patterns of community diversity in microbial ecology. In DGGE/TGGE, 16S rRNA gene fragments are separated on the basis of differences in their melting behaviour resulting in a pattern of bands on a gel (Muyzer & Smalla, 1998). Theoretically, each band represents a unique sequence and therefore a unique species (Powell et al. 2003). In T-RFLP, fluorescently labelled PCR products are digested with restriction enzymes and separated using automated sequencing technology. T-RFLP offers some important advantages over other fingerprint techniques, its resolution is higher and direct reference can be made to the 16S rRNA gene sequence database (Tiedje et al. 1999; Marsh et al. 2000). The application of FISH to microbial systems provides a way to detect and enumerate microorganisms in natural systems without culturing (eg. Giovannoni et al. 1988; Delong et al. 1989; Amann et al. 1990, 1991). FISH is a technique whereby fluorescently labelled DNA probes are annealed to a target sequence in nucleic acids of fixed cells. Probes have been used capable of identifying bacteria at varying levels of taxonomic hierarchy.

1.1.2.3 Ecological Function

By phylogenetically aligning an organism to its next nearest cultivated relative, we may shed light on the metabolic and physiological processes that are occurring (Pace, 1997). However caution is advised when considering the results of these studies as comparisons can only be

13 1.1 Microbial Ecology made when there is a high degree of sequence similarity between the identified phylotypes and known cultivated species, although even closely related organisms can show distinct physiological differences (Achenbach & Coates, 2000). For many clone sequences, no closely related cultivated species are known and until recently, linking most 16S rRNA gene information to function and ecological processes was dependent on culturing studies. Relatively new nucleic acid-based techniques, such as stable isotope probing (Radajewski et al. 2000) and bromodeoxyuridine labelling (Urbach et al. 1999), are beginning to emerge in the literature, allowing specific microbial processes and functions to be related to individual members of microbial communities in a cultivation-independent manner (Roling & van Verseveld, 2002).

These techniques rely on the synthesis of labelled DNA by microorganisms that grow in response to a specific stimulus and the subsequent separation of this labelled DNA from the pool of total DNA.

The use of biomarkers in combination with stable isotope analysis (eg. 13C) is one example of these relatively new culture-independent approaches to function analysis in microbial ecology. Biomarkers are compounds that have a biological specificity in that they are produced only by a limited group of organisms (eg. fatty acids, ether lipids). Natural abundance isotope ratios of biomarkers can be used to study organic matter sources utilised by microbes in complex ecosystems and for identifying specific groups of bacteria like methanotrophs

(Boschker & Middelburg, 2002). Addition of labelled substrates in combination with biomarker analysis enables direct identification of microbes involved in specific processes and also allows for the incorporation of bacteria into food web studies (Boschker & Niddelburg, 2002). Similarly,

FISH performed with rRNA-targeted oligonucleotide probes and microautoradiography can be used to analyse structure and function of bacterial communities. Lee et al. (1999) demonstrated the potential of this method by visualising the uptake of organic and inorganic radiolabelled substrates in probe-defined microbial populations.

To understand the role of a microorganism in a geochemical process, detection and identification of the microorganism in an environment in which the process is occurring is essential. Although demonstrating the presence of an organism in an environment where the

14 1.1 Microbial Ecology process is occurring does not mean the detected organism is important in the process of interest

(Siering, 1998). One ultimately needs to correlate the distribution and abundance of the organisms with the presence of the activity and the presence of any genes and gene products

(functional genes) involved in the process. If functional genes known to be involved in a particular process have been identified, isolated, characterised and sequenced, it is possible to use this information to develop PCR primers for amplifying the gene of interest from indigenous bacteria in natural samples. Hutchens et al. (2004) used DNA-based stable isotope probing and functional gene analysis of groundwater and mat material from Movile Cave to identify methane-assimilating populations and results suggest that aerobic methanotrophs

(Methylomonas, Methylococcus, Methylocystis/Methylosinus strains) actively convert CH4 into complex organic compounds and thus help sustain a diverse community of microbes in this closed ecosystem. This richness of methanotrophs was not revealed by RFLP analysis of the 16S rRNA gene clone library alone, demonstrating the benefits of constructing both 16S rRNA gene and functional gene libraries (Hutchens et al. 2004). Probing also increased already existing knowledge of microbial diversity in Movile Cave to include relatives of the cultivated and uncultivated members of the alpha, beta and gamma Proteobacteria, members of the

Acidobacterium division.

Amplifying and sequencing functional genes from organisms present in environmental samples allows us to investigate the distribution, evolutionary relationships, and diversity of functionally analogous genes (Siering, 1998). To prove a gene of interest is responsible for a process you must be able to detect expression of the gene in situ and correlate changes in gene expression with changes in the associated activity, for example detecting and quantifying the presence of particular messenger RNA (mRNA). This is often challenging due to the low quantities and very short lifespan of mRNA. Furthermore, gene expression studies require prior information, including transcript size and stability as well as expected levels of transcript present, which is not always available (Siering, 1998). Recent advances to increase detection sensitivities of gene expression rely on a form of PCR known as reverse transcriptase-PCR (RT­

PCR). Reverse transcriptase is used to synthesise a single stranded DNA copy (cDNA) of the

15 1.1 Microbial Ecology RNA template then the complementary strand of the cDNA is synthesised and the double­

stranded DNA molecule is subsequently amplified by normal PCR amplification.

1.1.3 Limitations of Methods

rRNA gene surveys have enormously extended the boundaries of microbial diversity,

but caution should be exercised when relying entirely on such an approach. In a detailed

culture-dependent survey of bacterial diversity in a wide range of deep-sea sediments, Li et al.

(1999) isolated 75 different actinomycetes; however very few actinomycete sequences were

cloned from these same samples in a later study (Colquhoun et al. 1998a,b, 2000).

The isolation of members of complex microbial communities as cultures also has

significant advantages over culture-independent molecular approaches given the inability to

identify with certainty the ecological, metabolic or physiological potential from novel molecular

sequence data (Atalan et al. 2000). It is most probable that the inability of microbiologists to

culture the majority of microbes in the laboratory results from the use of cultivation media that

does not resemble natural conditions or perhaps that some strains are interdependent (Wagner

et al. 1993). There is a trend emerging amongst microbial ecologists to continue to develop new

culture methods and media to attempt to cultivate novel taxa from so-called "unculturable"

groups of bacteria. In particular, Sait et al. (2002) and Joseph et al. (2003) had great success

culturing from Australian soils numerous phylogenetically novel microbes (the "Ellin" isolates)

belonging to previously uncultured groups using relatively simple cultivation methods.

Regardless, it is indisputable that culture-independent studies based on obtaining 16S rRNA

genes directly from the environment by broad-specificity primer PCR and cloning have greatly

improved our understanding of microbial diversity.

PCR-based surveys also have a number of recognised, inherent limitations. The quality

of extracted nucleic acids may be compromised by problems of shearing, degradation due to the

presence of contaminating nucleases, or contamination with humics or other substances known to inhibit subsequent molecular biological manipulations. Techniques must be optimised for

16 1.1 Microbial Ecology each type of environmental sample. Unfortunately, most methods for the extraction of nucleic acids from environmental samples lack a quantitative component; little data exists on the efficiencies of bacterial lysis and how these lysis efficiencies are affected by the complex matrix of biological and non-biological material within different sample types (Siering, 1998).

Unfortunately PCR does not necessarily occur in an accurate and unbiased fashion. A primary concern in amplifying 16S rRNA genes from mixed samples is the formation of chimeric sequences from the artifactual joining of 16S rRNA gene sequences of two organisms

(Liesack et al. 1991; Kopczynski et al. 1994) or from distinct copies of rRNA genes within the genome of a single organism (Wang & Wang, 1997). Such chimeric sequences occur at variable frequencies ranging from4.l-20% (Robison-Cox et al.1995) to 8.8-32% (Wang & Wang, 1997) and, therefore, should not be ignored. There are computational methods available to detect these artefacts (Robison-Cox et al. 1995; Komatsoulis & Waterman, 1997; Maidek et al. 1997), although all methods fail to detect some chimeras, especially those from closely related sequences, or misclassify non-chimeras as being chimeric. Hugenholtz & Hubert (2003) found during a recent collation within the public databases that, despite precautions taken, a surprising number of chimeric 16S rRNA gene sequences from molecular phylogenetic surveys were detectable.

However, by being vigilant and using several available methods rather than a single method, such inaccuracies can be decreased.

A separate issue is PCR bias, that genes are not equally amplified from all organisms

(Reysenbach et al. 1992; Suzuki & Giovannoni, 1996). This is one of the major drawbacks to developing quantitative PCR methods. Template bias is sometimes due to variable energetics in primer annealing and DNA denaturation due to G+C content in the template or primer DNA, in other instances causes for bias have not been identified (Suzuki & Giovannoni, 1996). Genome size and the number of different copies of rRNA genes within a given genome have also been shown to result in differential amplification of rRNA genes from mixed community DNA

(Farrelly et al. 1995). These parameters are unknown for the majority of organisms present in a given sample, thus Farrelly et al. (1995) contended that it is impossible to accurately quantify compositions of microbial communities by analysing clone libraries from amplified 16S rRNA

17 1.1 Microbial Ecology genes. Clone library analysis provides useful phylogenetic information that is reflective of community composition and relative distributions of organisms. However, small sample sizes prevent adequate representation of microbial community phylotypes because of cost and labour limitations. Community fingerprinting methods can alleviate these issues.

Although useful for quick comparisons of multiple communities, the drawbacks to fingerprint-based methods include a lack of resolution provided by gel-based separation and also difficulty in assigning phylogenetic information to the complex banding patterns that are usually obtained. With fingerprinting techniques, phylogenetic inference is most effective when only a single bacterial division or smaller group is addressed and is far less useful when the entire bacterial community is profiled (Dunbar et al. 2001). A combination of the two methods, fingerprinting and detailed clone analysis would be a more comprehensive way to study community composition.

18 1.2 Caves

1.2 Caves

Spaces below the Earth's surface range in size from microfissures to hundreds of kilometres in length and theoretically most have no natural human-accessible entrances (Curl,

1966 in Northup & Lavoie, 2001). A cave is defined as any natural space below the surface that

extends beyond the twilight zone and that is accessible to humans (Hill & Forti, 1986). Caves can

be classified in several ways, particularly by the type of rock and method of formation (Palmer,

1991). The most common types of caves are those formed in carbonate rocks. Other types of

caves are usually limited in extent and include those in gypsum, granite, quartz and sandstone.

1.2.1 Speleogenesis: Cave Fonnation

The birth of a cave system is referred to as speleogenesis (Ford & Cullingford, 1976). The

gradual solution of carbonate rocks, usually taking several millions of years, results in a wide

spectrum of landforms, collectively known as "karst" and caves are one of the most common

examples of this process. Carbonate rocks, such as limestone, are derived from the accumulation

of marine organisms (shells, corals etc) and as sediments on the sea floor. These marine

sediments consolidate over a long period of time and may be subsequently uplifted forming parts of the landmass of many regions of the world. Carbonate rocks contain carbonate minerals such as calcium carbonate (CaC03), often enriched with magnesium or iron and that are easily

dissolved by acids, even very weak solutions of acid.

Dissolution processes in carbonate rocks are due to the natural action of water. It occurs

as: i) surface water run off, flowing over impervious cap rock that lies above the more porous carbonate rock then flowing into carbonate, (swallet); ii) from a surface stream draining another rock surface further upstream, then entering the carbonate rock, (streamsink); or iii) rain water seeping through forest mulch and soils into the carbonate rock below (percolation water) (Ford

& Cullingford, 1976). These "charged" or "aggressive" waters are slightly acidic and penetrate

through points of weakness in the rock (eg. cracks, joints, bedding planes). Run off water or

19 1.2 Caves stream water also has a forceful action of erosion, corrosion and abrasion due to gravity, water mass or volume, and its sediment load of fine sands or gravels, which increases the magnitude of the dissolution process. The effect of seepage or percolating water is also ~ided by a number of factors. Rainwater contains dissolved carbon dioxide (C02) from the atmosphere forming a weak carbonic acid. This acidity is further strengthened by absorption of C02 from microbes and various humic or tannic acids from plant matter in the soil. Sulphuric acid sometimes derived from presence of sulphides in the soils, limestone or dolomite adds to the acidity of the water.

As the acidic water reaches the water table, it stays in contact with the carbonate causing further

dissolution of CaC03. This process is referred to as carbonic acid-driven speleogenesis.

Limestone caves may also be derived from a second process referred to as sulfuric acid-driven speleogenesis. Hydrogen sulfide rises along fissures until it encounters the oxygenated zone and forms sulfuric acid that dissolves the surrounding carbonate rock (Hill, 1990).

1.2.2 Speleothems: Cave Decoration

A cave, at constant temperature and invaded by percolating solutions carrying various substances, forms an excellent environment for the slow deposition of minerals (Ford &

Cullingford, 1976). One of the most commonly known aspects of caves is their visual beauty, due to their natural, internal formations, often referred to as cave decoration. These formations are secondary mineral deposits on the ceiling, floor and walls of a cave and are called

"speleothems". Most caves have enough openings to allow air movement, which evaporates some of the moisture and allows the precipitation of carbonate minerals from the seeping waters to form speleothems. Their creation depends on a number of factors: i) amount of seepage waters entering the ground above the cave, ii) type of rocks in and around the cave, iii) type of dissolved materials contained in the water as it enters the cave, and iv) the cave environment,

(eg. amount of moisture in the air, amount of air flow through the cave, cave temperature).

Formations are precipitated very slowly; it may take one hundred to one hundred and fifty years to form 2.5 cm of material and the slow growth and nearly constant conditions in

20 1.2 Caves caves results in these mineral deposits displaying spectacular crystal development (Ford &

Cullingford, 1976). The colouration of speleothems varies depending on the mineral composition of the carbonate rocks (eg. white or cream for almost pure CaC03, to yellowish or dark brown due to the presence of limonite, or red/ orange hues from dissolved iron, or blue hues from manganese). The colour variations and the various crystal configurations create the beautiful wonderland of this subterranean world.

Hill & Forti (1986) recognised 38 "official" speleothem types, with numerous subtypes and varieties, (Eg. stalactites, stalagmites, flowstones, rimstone pools and moonmilk) and described over 250 different minerals found in caves. Of special interest is moonmilk, a widely distributed, secondary formation and refers to the very hydrated white spongy /pasty or powdery masses found coating walls and speleothems in caves. It is composed of several carbonate minerals, predominately calcite. The wet pasty forms of moonmilk are so striking that some special explanation for their origin seems to be necessary, since calcite in cave environments usually has a completely different habit, hard and crystalline (Ford & Cullingford,

1976).

1.2.3 Cave Environment

Cave environments are strongly buffered against daily, seasonal and long-term climate changes occurring on the surface providing stable, sheltered and moist refuges for organisms.

The terrestrial cave environment is strongly zonal, with four major zones recognised; entrance, twilight, transition, and deep zone. The entrance zone is where the surface and underground environments meet. Beyond the entrance is the twilight zone where light still penetrates but progressively diminishes to zero. The transition zone is completely dark but the environmental effects from the surface are still felt. In the deep zone, environmental conditions are relatively stable, with fairly constant air and water temperatures (approximately the mean annual surface temperature) and the relative humidity near saturation resulting in an extremely low rate of

21 1.2 Caves evaporation (Barr & Holsinger, 1985 in Eberhard, 1999). Note that conditions may be less stable surrounding active, surface-fed streamways or passages near internal cave entrances.

The extent of the different zones depends on the size, shape and location of the entrance(s), on the-configuration of the cave passages and on the subterranean water/moisture supply (Howarth, 1988). The boundary between the transition and deep zones can be dynamic, changing on a seasonal or even daily basis, as air is pushed into, and pulled out of caves in response to changes in air density related to temperature and barometric fluctuations on the surface (Howarth, 1980). In temperate regions during summer, it is usually warmer outside the caves than inside, whereas in winter the reverse is true, generally resulting in a net movement of water vapour into caves during summer and out of caves during winter. Unlike the earth's surface, caves are not subject to the same weathering processes so what is found inside them often represents a different "snapshot" of the earth's history than would otherwise be available from the surface (http://www.speleonics.com.au; maintained by J. Rowling).

1.2.4 Speleology: Cave Study

The study of caves is called "speleology", and the study of life forms in caves,

''biospeleology". The main focus of biospeleologists is the deep, dark zone, also referred to as the hypogean environment, due to the highly specialised organisms found there. Hypogean environments are not restricted to caves, but include any system of crevices and fissures deeper than the soil layer. In caves, the hypogean domain is most conveniently open to study by man.

The hypogean domain may also be artificially penetrated for study particularly by mines and wells, both of which often yield hypogean organisms (Ford & Cullingford, 1976). These ecosystems are exposed to extreme environmental stresses and may be based on inorganic energy sources rather than sunlight. The limiting environmental characteristics of caves, little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate, provide ecological niches for highly specialised organisms. Historically, macroscopic life was the primary source of interest for study in caves. However recently biospeleologists have turned

22 1.2 Caves their attention to the microscopic life in these systems, revealing unique microbial ecosystems

(eg. Cunningham et al. 1995; Sarbu et al. 1996; Jones 2001; Holmes et al. 2001; Schabereiter­

Gurtner et al. 2002; Northup et al. 2003; Barton et al. 2004).

23 1.3 Microbial Biodiversity and Ecology of Caves

1.3 Microbial Biodiversity and Ecology of Caves

Caves are severely resource limited due to the absence of light that precludes primary production of organic material by photosynthetic organisms (Northup & Lavoie, 2001). Even so, microorganisms are widely distributed in caves and include bacteria, archaea, yeasts, fungi, and algae. Researchers proposed that the role of microbes in caves is to serve as a food source for higher trophic levels (eg. Dickson, 1979); however it was typically believed that microbes could not provide adequate energy to support a large and diverse ecosystem. In contrast, the work of

Sarbu et al. (1996) in Movile Cave, Romania, and Vlasceanu et al. (2000) in Frasassi Caves, Italy, suggest that chemoautotrophic, sulfur-based microbial communities can generate enough energy as primary producers to sustain complex cave ecosystems. These caves receive little or no surface-derived organic material, but instead microbially reduced sulfur compounds in the cave waters provide the energy for carbon dioxide fixation (Mattison et al. 1998). The work in

Movile Cave provided evidence of the first terrestrial microbial community known to be chemoautotrophically-based (Sarbu et al. 1996).

Culture-independent 165 rRNA gene sequence analyses have opened the way to study bacterial communities in environmental samples without prior cultivation and have revealed a significantly broader diversity than culture-based studies in many environments over the last 25 years (Amann et al. 1995; Head et al. 1998; Hugenholtz et al. 1998). Nevertheless, to date our knowledge of bacterial communities in caves has been largely due to culture studies (eg. Groth et al. 1999a; Laiz et al. 1999). As discussed in Section 1.1.2.3, phylogenetic analyses can be used to hint at the ecological functions of uncultivated phylotypes obtained from molecular analyses.

The recent influx of molecular analyses of cave microhabitats (Eg. Holmes et al. 2001;

Schabereiter-Gurtner et al. 2002; Northup et al. 2003; Barton et al. 2004; Chelius & Moore, 2004;

Schabereiter-Gurtner et al. 2004) have attempted to do just this; elucidating the roles of bacteria in caves, how they survive, interact with, and affect, their environment.

24 1.3 Microbial Biodiversity and Ecology of Caves 1.3.1 Chemolithoautotrophic Systems

In cave ecosystems with little or no exogenous organic input, the rich variety of redox interfaces allows primary growth of chemolithotrophic (eg. ammonium-, nitrite-, sulfur-,

manganese- or iron- oxidising) bacteria (Northup & Lavoie, 2001). Several studies of

chemolithotrophic communities have been reported in the literature and have demonstrated that

these bacteria play an important role in some cave ecosystems, acting as primary producers and

supporting growth of heterotrophic microbes (eg. Sarbu et al. 1996). These subsurface microbial

communities are based on chemolithoautotrophic energy processing where life does not depend

directly upon energy and organic carbon from photosynthesis (Stevens & McKinley, 1995).

1.3.1.1 Sulfur-based Systems

Caves containing hydrogen sulfide-rich springs represent less than 10% of all known

caves globally (Summers Engel et al. 2003). These caves serve as access points into sulfidic

groundwater aquifers, typically associated with geothermal regions and oil-field basins, which play an important role in global sulfur cycling. The microbial communities colonising sulfidic cave habitats are of particular interest due to their chemolithoautotrophic metabolism that can

sustain a high biomass and rich complex ecosystems in the subsurface (Sarbu et al. 1996; Angert et al. 1998; Hose, 1999) and their geomicrobiological impact, for example sulphuric acid-driven

speleogenesis (Engel et al. 2001; Vlasceanu et al. 2000).

Frasassi Cave, Italy, and Cueva de Villa Luz, Mexico, are sulfidic cave systems where sulfuric acid drips from the walls and deadly levels of hydrogen sulfide and carbon monoxide are emitted from springs. Yet amidst these hostile conditions a rich and diverse ecosystem of invertebrates and microorganisms are alive and well. Biofilms consisting of extreme acidophiles grow on thick crusts of gypsum and elemental sulfur on the cave walls. Clone library analysis of the Frasassi biofilm revealed at least 2 strains belonging to the genera Thiobacillus and

Sulfobacillus (Vlasceanu et al. 2000). An acid producing strain of Thiobacillus was also cultivated from the Frasassi biofilm. A defining feature of members of the Thiobacillus genus is their ability

25 1.3 Microbial Biodiversity and Ecology of Caves to gain energy from the oxidation of reduced sulfur compounds and resulting production of

sulfuric acid. Stable carbon isotope analysis revealed that the wall biofilms in Frasassi Cave are

isotopically light. Terrestrial isopods living on the cave walls showed peculiar isotopic values

markedly different from the rest of the invertebrates inhabiting the cave, implying that they are

feeding on these biofilms (Vlasceanu et al. 2000). These results imply that the cave food-web is

based on organic matter produced chemoautotrophically in situ by sulfur-oxidising microbes

forming mats that cover the bottom and the mudbanks of the streams and the walls of the cave,

similar to the microbial mats of the sulfidic springs of Movile Cave, Romania (Vlasceanu et al.

2000).

Microbially generated acid formed in the Frasassi biofilms diffuses through the gypsum

to the carbonate surface or drips from the tips of the microbial biofilms onto exposed carbonate

surfaces, causing rock dissolution (Vlasceanu et al. 2000). Summers-Engel et al. (2001)

investigated microbial diversity in mats from hydrogen sulfide rich waters and cave wall

biofilms in Cesspool Cave, Virginia, and pure cultures of Thiobacillus spp. isolated from this mat,

demonstrated the ability to corrode CaC03 (Summers-Engel et al. 2001). Corrosion of CaC03

substrata causes subsequent gypsum precipitation. Substrate dissolution can be beneficial to

microbes due to the release of nutrients such as nitrogen and phosphorus in oligotrophic

habitats (Rogers et al. 1998), but rock dissolution can also be detrimental in the case of carbonates

because of pH fluctuations, and in other rocks due to the release of toxic compounds, including

aluminium or trace elements (Engel et al. 2001).

The formation of caves in limestone bedrock was traditionally considered to be driven

by carbonic acid dissolution of carbonate, as discussed in Section 1.2.1. In contrast the formation

of Carlsbad Cavern and Lechuguilla Cave, New Mexico, and Movile Cave, Romania, is

inconsistent with this model of speleogenesis. Hill (1990) suggested that in caves where

hydrogen sulfide-rich waters are present, the production and activity of sulfuric acid might be

the primary cause of carbonate dissolution. Initially it was assumed to be a nonbiological

process, the sulfuric acid resulting from the chemical oxidation of hydrogen sulfide. Parker &

Jackson (1965), however, presented evidence that sulfuric acid production may be mediated by

26 1.3 Microbial Biodiversity and Ecology of Caves Thiobacillus spp. Since then, several studies have confirmed the significant role of acid produced by sulfur-oxidising bacteria in the dissolution of limestone suggesting that the colonisation and metabolic activity of these bacteria may be enhancing cave enlargement (Engel et al. 2001;

Vlasceanu et al. 2000).

1.3.1.2 Iron, Manganese, Nitrite, and other Systems

Microorganisms living at the interface between the host rock and cave passages can utilise reduced compounds in the host rock. Caves formed by the dissolution of limestone by carbonic acid are often enriched in iron, manganese and nitrogen and studies have yielded circumstantial evidence for chemolithoautotrophy by iron-, manganese- and nitrogen- oxidisers in these systems (Northup et al. 2000, 2003; Holmes et al. 2001). Unusual aquatic formations, mantles of mucus and biological material associated with crystalline material, in submerged passages of the Nullabor Caves, Australia contain a high proportion of phylogenetically novel sequence types and a high relative abundance (approximately 12%), of Nitrospira relatives, showing most similarity to autotrophic nitrite-oxidising bacteria (Nitrospira moscoviensis).

Holmes et al. (2001) concluded that this community structure, the presence of nitrite in the water, and the apparent absence of aquatic macrofauna, indicate biochemically novel, chemoautotrophic communities dependant on nitrite oxidation.

Lechuguilla Cave, New Mexico, is an immense ancient cave in near pristine condition, an extremely low nutrient environment with, however, sulfur, iron, and manganese deposits harbouring diverse microbial life (Northup et al. 2003). 16S rRNA gene clone analysis of corrosion residues (ferromanganese deposits) showed the presence of known iron- and manganese- oxidising/reducing bacteria including phylotypes of the genera, Hyphomicrobium,

Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea (Northup et al. 2003). Black ferromanganese sediments in Vantului Cave, Romania, contain Hyphomicrobium spp.,

Pedomicrobium fusiforme and Pedomicrobium mangancum, known to mediate the oxidation and precipitation of manganese (Manolache & Onac, 2000). Northup et al. (2003) suggested that these diverse communities of microbes inhabiting ferromanganese deposits seem to exist by utilising

27 1.3 Microbial Biodiversity and Ecology of Caves manganese and iron from the bedrock and that the ferromanganese deposits represent, at least in part, the end-product of microbially assisted dissolution and leaching of the underlying host carbonate, and enrichment of iron and manganese through microbial oxidation (Northup et al.

2000).

Literature on oligotrophic cave communities subsisting in regions of nutrient scarcity is still sparse and the majority of these investigations have concentrated on communities sustained by a specific and measurable energy input, whether from sulfide, nitrite or surface organic input

(eg. Sarbu et al. 1996; Angert et al. 1998; Holmes et al. 2001). Barton et al. (2004) investigated microbial diversity in Fairy Cave, Colorado, looking at a wall community in the absence of observable energy sources. Their studies revealed a larger diversity in an oligotrophic environment than originally thought (phylotypes from 4 different divisions, Proteobacteria,

Actinobacteria, Cytophagales and the low G+C Gram-positive bacteria). The limestone bedrock of

Fairy Cave is almost pure CaC03, (>97.5%) with no significant reduced metal compounds available to act as electron donors and any metal ions that are present in the cave system were likely deposited by the rich mineral waters that formed the cave system (Barton et al. 2004).

Metabolic analyses suggested that the community subsists using a complex metabolic network with input from trace organics within the environment or fixation of atmospheric gases using lithotrophic metabolism (Barton et al. 2004).

A common theme was observed in cultivated relatives of the cloned phylotypes from

Fairy Cave, the fixation of atmospheric gases or the use of aromatic carbon compounds. The source of atmospheric gases is obvious, while the potential carbon sources may be the inorganic constituents of water filtering into the cave system. Previous research has suggested that cave waters contain dissolved organic matter from the soil, primarily phenolic compounds and lignin

(Saiz-Jimenez & Hermosin, 1999). These compounds can be utilised as carbon sources by many of the species related to those found in Fairy Cave. Similar mechanisms of lithotrophy have been suggested for other cave systems (eg. Cunningham et al. 1995). Northup et al. (2000) also suggested that reduced metals, such as magnesium and iron, within the limestone matrix of

28 1.3 Microbial Biodiversity and Ecology of Caves Lechuguilla Cave provide a sufficient source of electron donors for growth, which may further require the presence of atmospheric organic molecules as a carbon source.

In contrast to the molecular evidence, generally few chemolithoautotrophic bacteria in caves have been detected by cultivation as well as by PCR-based studies (Sarbu et al. 1996;

Vlasceanu et al. 2000; Engel et al. 2001; Holmes et al. 2001). As discussed in Section 1.3.1, molecular analyses revealed unexpected dominance of mostly uncultured groups, (eg. Epsilon

Proteobacteria in sulfidic springs of Lower Kane and Parker Caves). The majority of chemoautotrophic species isolated from caves belong to, the sulphur- and sulphide-oxidising genera, (Thiobacillus, Thiosphaera, Thiothrix, Thiomicrospira, Beggiatoa, Achromatium, Sulfobacillus and Thioalcalovibrio); the sulphate-reducing Desulfovibrio sp.; the iron-oxidising Leptospirrillum ferrooxidans and Thiobacillus ferrooxidans; the manganese- and iron-oxidising genus Leptothrix and nitrifiers such as Nitrobacter sp. (Schabereiter-Gurtner et al. 2002). Culture-independent analyses of Fairy Cave revealed a community distribution of phylotypes unique from previous observations in oligotrophic caves using cultivation, suggesting that many of the species identified are sufficiently adapted to the oligotrophic lifestyle and thus remain resistant to cultivation using standard techniques (Barton et al. 2004).

1.3.2 Heterotrophic Systems

Cave microbial communities usually rely on allochthonous input of organic matter transported from the surface (Groth et al. 1999a). In caves, animals and visitors can provide large amounts of organic input facilitating heterotrophic life (Hose et al. 2000; Groth & Saiz-Jimenez,

1999). Culture-dependent studies have focused on heterotrophic caves with allochtonous input of organic matter demonstrating that heterotrophic bacteria often dominate these communities

(Groth & Saiz-Jimenez, 1999). Many microbes identified from deep caves are similar to surface forms and are probably transported into caves by water, air, sediment and animals (Saiz­

Jimenez 2001; Schabereiter-Gurtner et al. 2002a, b). Actinomycetes are the most abundant

29 1.3 Microbial Biodiversity and Ecology of Caves heterotrophic Gram-positive bacteria to be isolated from these caves particularly streptomycete, nocardioform and coryneform actinomycetes (Groth et al. 1999a).

Organic input may also be dissolved in the seepage/ dripping waters or as particulate organic matter carried in by active or periodic flooding of a subterranean streamway

(Schabereiter et al. 2002). High sulphate and nitrate concentrations have been found in dripping waters in Tito Bustillo and other Spanish and Italian caves (Hoyos et al. 1999) which, in addition to the concentrations of iron, manganese and other elements found in cave rocks, supports heterotrophic bacteria involved in the nitrogen, sulphur, iron and manganese cycles. Laiz et al.

(1999) investigated the microbial diversity of dripping waters of Altamira Cave, Spain. Water communities were not dominated by actinomycetes but contained low proportions of Gram­ positive bacteria, and were mainly composed of Gram-negative rods and cocci (Enterobacteriaceae and Vibrionaceae; genera Aeromonas and Acinetobacter). Compounding this, in an earlier study of dripping waters in Altamira Cave carried out by Somavilla et al. (1978) Bacillus and Pseudomonas appeared to be the most abundant genera, followed by Flavobacterium and Erwinia. In comparison, isolations from ceiling rock of Altamira Cave resulted mainly in Gram-positive

Streptomyces spp. The absence of culturable actinomycetes in dripping waters agrees with the observations of Kolbel-Boelke et al. (1988). They found very few actinomycetes in 60 water and sediment samples clearly demonstrating that dripping water communities are very different to those of cave rock though both are heterotrophic based systems.

Wind Cave, South Dakota, is a heterotrophic detritus-based limestone cave. Clone library analysis by Chelius & Moore (2004) illustrated that Gamma Proteobacteria and

Acidobacterza predominated water-saturated sediments in the dark zone. Furthermore, most of the microbial sequences were not related to known chemolithoautotrophs, therefore it was concluded that this particular community is likely detritus-based, where allochthonous energy and carbon are transported into the cave by infiltrating waters. Although some clones resembled sequences from other caves, they found that no cave-specific bacterial community was evident.

Clones mostly resembled those from soil, freshwater, plant associated and polluted environments (Chelius & Moore, 2004). Conversely, culture studies of the same sediments from

30 1.3 Microbial Biodiversity and Ecology of Caves Wind Cave produced representatives of only the actinomycetes and Proteobacteria (Alpha, Beta and Gamma) though clone analysis indicated that these were relatively minor components of the microbial community (Chelius & Moore, 2004). Most isolates were related to other cultivated members and sequences retrieved from soil and various polluted environments. It is important to note that Wind Cave is also a show cave, impacted by humans and lighted for tours, but this study still represents baseline data. Although in general molecular analyses reveal them as relatively minor representatives of cave communities, actinomycetes are still the most dominant group of bacteria isolated from caves (Schabereiter-Gurtner et al. 2002; Chelius & Moore, 2004).

1.3.3 Actinomycetes in Caves

Results of studies in caves of China, Korea, Northern Spain, and Southern Italy have demonstrated that actinomycetes are not only the most abundant bacteria isolated from these caves, but also reveal a great taxonomic diversity (Groth et al. 1999a,b; Groth & Saiz-Jimenez,

1999). Several new species of actinomycetes have been described from hypogean environments

(Lee et al. 2000a,b,c; Lee et al. 2001) including three new genera, Knoellia sinensis gen. nov., sp. nov. and Knoellia subterranean sp. nov, and Beutenbergia cavernae gen. nov., sp. nov., isolated from sediment sampled from Reed Flute Cave, China (Groth et al. 2002, 1999b); Hongia koreensis gen. nov., sp. nov., isolated from sediment in a gold mine cave of Korea (Lee et al. 2000). Three novel species were also described from the gold mine cave in Korea, Pseudonocardia kongjuensis sp. nov. and Saccharothrix violacea sp. nov., and S. albidocapillata comb. nov (Lee et al. 2000, 2001).

However, little has been published about the cave environments that these novel species have been described from.

Caves are not uniform environments in terms of geological and geochemical characteristics, as they can vary from one to the other, eg. rock type, method of formation, length, depth, number of openings to the surface, presence or absence of active streamways, degree of impact by human visitation etc. Furthermore, on a smaller scale, various microhabitats, with vast differences in community structure can exist within caves. It seems

31 1.3 Microbial Biodiversity and Ecology of Caves fairly widely accepted that dry cave substrate typically yields a higher proportion of actinomycetes than does dripping water and wet sediment (Kolbel-Boelke et al. 1988; Laiz et al.

1999). Somavilla et al. (1978) did not culture actinomycetes from the air of Altamira and La

Pasiega Caves, whereas Arroyo & Arroyo (1996) found actinomycetes from contact plates from the floor, walls, and ceilings of the same cave.

Limestone caves and lava tube caves often contain wonderful displays of filamentous actinomycetes that may cover entire ceilings and walls of caves giving a 'silvered' appearance.

Probably many of the discrete lichen-like colonies frequently noted on walls and formations in the dark zone may be actinomycetes of the genus Streptomyces since they often have the powdery appearance and characteristic earthy odour common to cultures of this genus. It has also been suggested that the abundant Streptomyces in caves is probably responsible for the earthy smell of caving (Caumartin, 1963 in Ford & Cullingford, 1976). Streptomyces and Nocardia are the most common, and abundant, groups isolated from caves (Arroyo & Arroyo, 1996).

Streptomyces species are particularly abundant though this may be due to their easy growth in the laboratory.

The majority of the work on actinomycetes in hypogean environments has been conducted in Altamira, Tito Bustillo, La Garma, and Llonin caves, Spain, and Grotta dei Cervi,

Italy all of which have spectacular galleries with paleolithic rock art paintings (Groth & Saiz­

Jimenez, 1999; Groth et al. 1999a, 2001; Laiz et al. 1999, 2000). Groth et al. (1999a) reviewed the growth of actinomycetes on the ceiling and walls in Altamira and Tito Bustillo caves isolating approximately 350 strains. Actinomycete growth was distributed all over the caves and could be observed on the active stalactites, on upper and lower parts of the rock wall and in the cave soils. Large parts of the cave's rock surfaces were covered by macroscopic colonies (1-2 mm) visible to the naked eye and direct isolation from these colonies resulted solely in Streptomyces xanthophaeus. However culture-independent DGGE analyses detected 14 separate bands representing other species, most of them closely related to uncultured bacteria affiliated with

Proteobacteria, Acidobacteria, Cytophaga-Flavobacteria-Bacteroides group (CFBs) and Actinobacteria

(Schabereiter-Gurtner et al. 2002).

32 1.3 Microbial Biodiversity and Ecology of Caves Samples of active stalactites, wall concretions and rocks from the walls and ceilings of

the galleries have been investigated and a high number of isolates obtained. Most abundant

were genera of the actinomycetes, particularly Streptomyces especially from rock walls and soils.

Other genera isolated included Nocardia, Nocardioides, Saccharothrix, Amycolatopsis,

Brevibacterium, Rhodococcus, Aureobacterium, and members of the family Micrococcaceae. However,

in stalactites, the most abundant species isolated belonged to the low G+C Gram-positive

bacteria of the genus Bacillus, although the most conspicuous and visible to the naked eye were

actinomycetes of the genera, Agromyces, Amycolatopsis, Arthrobacter, Nocardiopsis, Rhodococcus,

and Streptomyces (Groth et al. 2001). These microorganisms are able to colonise the bare rock

surfaces utilising organics in dripping water. Apart from published novel species from caves,

most other papers characterise cave strains to the genus level only as they use morphological

and biochemical means of identification rather than phylogeny. At present it is therefore

difficult to make comparisons at the species level between cave environments.

Culture-dependent studies have focused on typical heterotrophic microbes from the

surface and have mostly come from so called "show caves" open to the public and which are

heavily impacted by humans. There is an apparent correlation between the number of visitors

and diversity of bacteria. The higher the number of visitors the higher the diversity of isolated

strains, as indicated by the data obtained in Tito Bustillo and Altamira caves (Groth et al. 1999a).

Altamira Cave revealed a great taxonomic diversity with predominant isolates belonging to

Streptomyces, Nocardia, Nocardioides, Saccharothrix, Amycolatopsis, Brevibacterium, Rhodococcus,

Aureobacterium, and members of the family Micrococcaceae (Groth et al. 1999a). Caves with

restricted access, Llonin and La Garma, yielded lower diversity. This increasing diversity is likely associated with lighting, which promotes the growth of phototrophic microorganisms,

and also the introduction of organic matter by visitors into the ecosystem (AriflO & Saiz-Jimenez

1996). Thus one could argue that it is the public nature of these caves that tend to heterotrophy

dominated by actinomycetes, rather than it being a general trend in cave systems with

allochthonous input of organic matter. However, Grotta dei Cervi shows similar colonisation patterns to Altamira Cave, in spite of the fact that this cave was discovered more recently in 1970

33 1.3 Microbial Biodiversity and Ecology of Caves (91 years later than Altamira) and visitation is restricted to scientific purposes (as in Llonin and

La Carma), the biodiversity was surprisingly high. This is perhaps due to the appreciably high

input of organic matter present in Grotta dei Cervi in the form of bat guano, promoting

heterotrophy and dominance of actinomycetes.

The study of cultivated microbes in these caves has revealed only a minor and not very

representative proportion of the cave microbial populations. Gram-positive bacteria identified in

Llonin, La Carma, Altamira, and Tito Bustillo Caves by culture-independent techniques is

relatively low (<30 %), though Gram-positive bacteria, and in particular actinomycetes were the

dominating isolates obtained from cultivation (eg. Groth & Saiz-Jimenez, 1999; Schabereiter­

Gurtner et al. 2002). More recently DGGE community fingerprinting combined with

phylogenetic analyses used to investigate samples from paintings and surrounding rock in

Altamira and Tito Bustillo revealed greater taxonomic diversity detecting unknown and

unexpected bacterial groups, particularly the Proteobacteria, Acidobacteria division, CFBs,

actinomycetes, green non-sulfur bacteria and Planctomycetes. DGGE analysis of paintings in

Llonin and La Carma caves (Schabereiter-Gurtner et al. 2004) also illustrated a high biodiversity

of chemolithotrophic, as well as heterotrophic, bacteria; the most abundant groups found were

the Proteobacteria, actinomycetes and Acidobacteria. This data compared to results from Altamira

and Tito Bustillo caves revealed similarities in the bacterial community components, especially

in the high abundance of the Acidobacteria and Rhizobiaceae, and ammonia- and sulfur-oxidisers

(Schabereiter et al. 2002). Which is interesting in that Llonin and La Carma are restricted visitor

access for research purposes only whereas Altamira and Tito Bustillo are open to the public.

These studies have revealed diverse and unknown microbial colonisation on the paintings in

contrast to previous culture-dependant investigations.

In the past, the study of microbial communities and biogeochemical processes in hypogean environments is mainly related to the fact that microbes affect cultural heritage

properties that humans wish to protect (Groth & Saiz-Jimenez, 1999) and we owe much of our initial knowledge of cave microbiota to these studies. The role of actinomycetes in the

deterioration of paintings and frescoes in hypogean environments (not just caves, but crypts,

34 1.3 Microbial Biodiversity and Ecology of Caves tombs and underground churches) has been emphasised by many investigations (eg. Monte &

Ferrari, 1993; Groth & Saiz-Jimenez, 1999; Groth et al. 1999a). Actinomycetes are known to destroy wall paintings by the excretion of organic and inorganic metabolic products

(Schabereiter-Gurtner et al. 2004). The first actinomycetes identified as degraders of rock art were Streptomyces rectus fiexibilis, S. griseolus, S.cinereoruber, S.vinaceus, S.albus and Nocardia sp.

(Giacobini et al. 1988).

Though the role of actinomycetes in rock art is highly recognised, it is interesting that they haven't been isolated from works of art, except those located in hypogean environments

(eg. caves, crypts, grottos and tombs) (Giacobini et al. 1988). Atlanterra Shelter, Spain, contains rock art paintings made with iron oxides. The shelter is exposed to terrestrial environmental fluctuations. The bacteria isolated from Atlanterra Shelter seem to constitute a homogenous community with abundance of Bacillus strains, very different to actinomycete dominated communities found in rock art paintings from karstic hypogean environments (Groth & Saiz­

Jimenez, 1999; Laiz et al. 1999; Gonzalez et al. 1999). All isolated Bacillus strains were able to reduce hematite which is significant due to the fact that Fe(ill)-(hydr)oxides are the most abundant pigments in rock art. This work demonstrates that actinomycetes are not alone in their role as biodeteriogens of rock art, however they do seem to be the dominant group in hypogean environments, perhaps indicating favourable selective pressures in the cave environment.

A number of actinomycetes isolated from caves have the ability to produce various types of crystals. Studies in Altamira and Tito Bustillo Caves demonstrate that the host-rock, cave formations and rock art are coated by dense networks of bacteria, mainly actinomycetes and these bacteria can induce constructive (calcification, crystalline precipitates) and destructive

(irregular etching, spiky calcite) fabrics. Because of this ability it has been proposed that these bacteria and others are directly or indirectly involved in constructive biomineralisation processes in caves (Laiz et al. 1999; Barton et al. 2001; Canaveras et al. 2001; Groth et al. 2001;

Jones, 2001). Little is known concerning the distribution, population dynamics, growth rates and biogeochemical processes of actinomycetes in caves, in spite of the fact that they seem to constitute a significant part of the "culturable" microbial population of these habitats. A

35 1.3 Microbial Biodiversity and Ecology of Caves prerequisite for the study of the role of actinomycetes in biogeochemical processes is the isolation and identification of these organisms (Groth et al. 1999a).

1.3.3.1 Actinobacteria

Stackebrandt et al. (1997) proposed a new hierarchic classification system, Actinobacteria classis nov. for the actinomycete line of descent, wholly defined by the phylogenetic analysis of small subunit 165 rRNA gene sequences. The Actinobacteria is comprised of high-G+C content

Gram-positive bacteria with a common ancestry and includes Subclasses Acidimicrobidae,

Rubrobacteridae, Coriobacteridae, Sphaerobacteridae and Actinobacteridae. The Order Actinomycetales

(actinomycetes) is within the Subclass Actinobacteridae. It is important to note here that quite often the actinomycetes are referred to simply as Actinobacteria, which, although fundamentally correct, is misleading, as the Class Actinobacteria encompasses a broader range of taxa than the

Actinomycetales alone. For the purpose of this study the term actinomycete(s) will be used to describe only members of the Class Actinobacteria, Subclass Actinobacteridae, Order

Actinomycetales.

1.3.3.2 Actinomycetes

Actinomycetes are Gram-positive bacteria which form branching hyphae at some stage of their development and may produce a spore bearing mycelium (McCarthy & Williams, 1990).

They are aerobic saprophytes and are widely distributed in nature (Goodfellow & Williams,

1983) mainly found in soil where they manufacture enzymes which degrade complex molecules and play a major role in decomposition of organic matter (Lechevalier & Lechevalier, 1985).

These organisms are selected for in environments characterised by oligotrophic conditions, low

water activities and high concentrations of CaC03. Hyphal actinomycetes are typically slow growing and their spores can remain viable for a number of years in unfavourable conditions; the exact length of time for which they can survive is uncertain. Although predominantly soil bacteria, actinomycetes have been isolated from a wide variety of environments, including

36 1.3 Microbial Biodiversity and Ecology of Caves freshwater, lake sediments, rivers, streams, marine environments, salt marshes, fodder and related materials, and air (Loyd, 1969; Cross, 1981; Hirsch & McCann-McCormick, 1985; Labeda

& Shearer, 1990). Actinomycetes have also been isolated from extreme environments such as; ice, sediments and air in Antarctica, and, as discussed previously, rock surfaces and sediments in cave environments (Eg. Cameron et al. 1976; Groth et al. 1999a,b).

1.3.3.3 Actinomycete Taxonomy

Over 150 genera of actinomycetes have been isolated from soils. The exact composition and phylogenetic boundaries of the actinomycetes has remained open to question and modification due to continued development and application of new taxonomic classifications.

Early attempts at taxonomic classification of actinomycetes were based on morphological and pigmentation characteristics of the sporing bodies and substrate mycelia, which is a useful but arbitrary approach to classification and not based on the phylogenetic relationships between different species (Williams et al. 1983). Variation in biochemical and physiological properties were incorporated into actinomycete taxonomy, however these new data alone could not be used to devise a satisfactory phylogenetically based taxonomy (Embley & Stackebrandt, 1994).

The rich chemical, morphological and physiological diversity of phylogenetically closely related genera of actinomycetes makes the description of families and higher taxa so broad that they become meaningless for the description of the enclosed taxa (Stackebrandt et al. 1997).

The application of molecular techniques based on variations in nucleic acid sequences between different bacteria, especially 165 rRNA gene sequencing, has had a dramatic impact on actinomycete systematics. It was soon discovered that some morphological characteristics given greater weight in earlier studies, such as the ability to form spores, were not reliable in a phylogenetic system of classification (Stackebrandt et al. 1981). Almost any description based on morphology or physiology would have exceptions and actinomycete taxonomy now relies heavily on molecular comparisons (Ensign, 1992). The only phenetic characteristics shared by all members of the actinomycetes is a relatively high level of guanine (G) and cytosine (C) as a

37 1.3 Microbial Biodiversity and Ecology of Caves percentage of total DNA (>55%) (Goodfellow, 1989). Actinomycete taxonomy is still under

development and more taxonomic information needs to be collected in all fields in order to

develop a phylogenetic system of classification with confidence (Holloway, 1997). To determine

a phylogenetic classification of actinomycete which is both true and practical it is necessary to

employ a polyphasic approach, employing a combination of molecular, chemical and numerical

taxonomic methods (Murray et al. 1990).

1.3.3.4 Actinomycete Ecology

As soil bacteria, actinomycetes contribute significantly to the turnover of complex

biopolymers, such as lignocellulose, hemicellulose, pectin, keratin and chitin (Williams et al.

1984). Additionally nitrogen-fixing actinomycetes of the genus Frankia have one of the broadest

host ranges known, forming root nodule symbioses in more than 200 species of flowering plants

(Huss-Danell et al. 1997). Actinomycetes can be recovered from most soils in relatively high

numbers although this may not give an accurate picture of proportions of active bacteria in the

soil because most of the colonies are probably isolated from spores (Williams, 1978). Streptomyces

and Arthrobacter are ubiquitous in soil and are the most numerous of the actinomycetes

(Goodfellow & Williams, 1983). The next most common actinomycetes are, in descending order,

members of the genera Micromonospora, Actinoplanes, Actinomadura, and Nocardia (Lechevalier &

Lechevalier, 1985).

Although soil is the main habitat of the actinomycetes, they can be isolated from

humans, animals, plants, waste water, food products, stones, buildings and works of art (eg.

Groth & Saiz-Jimenez, 1999). Despite intensive studies there are still many gaps in our

knowledge of the role played by actinomycetes in soil processes (Goodfellow & Williams, 1983).

Caves are unique environments characterised by little or no light, low levels of organic nutrients, and a stable, but cool to cold, microclimate. Russell, (1990), hypothesised that it is not

necessary for a microbe to function at optimal rates as long as it can compete effectively in its

38 1.3 Microbial Biodiversity and Ecology of Caves particular environment. It may be quite advantageous for cave bacteria to metabolise submaximally and have long generation times in nutrient poor environments.

Actinomycetes are well known for their ability to grow on nutrient poor media

(Lechevalier & Lechevalier, 1985) and streptomycetes can exist for extended periods of time as arthrospores that germinate in the occasional presence of nutrients (Goodfellow & Williams,

1983). Low temperatures are not a limiting factor for actinomycete growth. Suzuki et al. (1997) described an obligately psychrophilic actinomycete (Cryobacterium psychrophilum), and Xu et al.

(1996) reported actinomycete populations in cool areas of China, with average temperatures of

5° C or below 0° C, where Streptomyces spp. constituted up to 97% and 83%, respectively, of the total heterotrophic count. Some were psychrophiles with an optimum growth temperature of

10-15° C. Groth & Saiz-Jimenez (1999) suggested that growth of actinomycetes in hypogean environments might result from the association of two factors: low temperatures and high relative humidity. These environmental conditions, together with nutrient availability and nature of organic matter are recognised to be important factors controlling the activity of actinomycetes in caves.

39 1.4 Geomicrobiology

1.4 Geomicrobiology

Geomicrobiology is the term given to studies of the microbe-mineral interface, including microbial weathering and sedimentation processes, microbial roles in formation and degradation of minerals, mineralisation of organic matter, subsurface microbiology, biogeochemical cycling of elements, and bioremediation. Microorganisms are important active and passive promotors of redox reactions that influence geological formations (Ehrlich, 1999).

There is extensive literature demonstrating the influence of microorganisms in mineral formation from non-cave environments for a wide variety of minerals including, carbonates, oxides, phosphates, sulfides, and silicates (Fortin et al. 1997). Bacteria may produce minerals as a result of growth. Cell walls have chemically reactive sites that bind dissolved mineral-forming elements allowing nucleation and growth of crystals from an oversaturated solution to occur

(Groth et al. 2001). Alternatively, mineral precipitation may result from metabolic activities of bacteria. Bacterial activity may simply trigger a change in solution chemistry that leads to oversaturation and mineral precipitation. In biological processes, oversaturation is considered an important prerequisite for the precipitation of minerals from solution (Fortin et al. 1997).

Although Gonzalez-Munoz et al. (1996), suggested that this is merely incidental and the critical point is the participation of cellular membranes in inducing nucleation. Caves can be used as experimental study systems for geomicrobiology, not because they are strange, but because they are simple and often locally abundant, allowing for replicate studies (Northup & Lavoie, 2001).

While geomicrobiology in general has received substantial interest in the last decade, one unresolved issue is the involvement of microbial activity in the dissolution of, or formation of speleothems in caves (Barton et al. 2001).

1.4.1 Geomicrobiology in Caves

Caves are nutrient-limited environments containing a variety of redox interfaces and they provide an accessible window into subsurface environments in which to study precipitation and dissolution processes and products (Northup & Lavoie, 2001). A variety of 40 1.4 Geomicrobiology precipitation and dissolution processes results in the deposition of carbonate speleothems, silicates, iron and manganese oxides, sulfur compounds and nitrites and the break down of limestone walls resulting in corrosion residues. Geomicrobiological activities in caves are no longer underestimated since studies have shown that bacterial metabolism can affect these mineral precipitation and dissolution processes (Cai\.averas et al. 2001; Northup & Lavoie (2001).

Studies of microorganisms in caves have been predominantly descriptive, as illustrated in

Section 1.3, with only a few experimental studies reported although increased interest in microbe-mineral interactions in caves is emerging.

Microbially influenced corrosion or dissolution of mineral surfaces can occur through mechanical attack, the secretion of enzymes, and organic and mineral acids (eg. Sulfuric acid).

Microbially mediated reactions can generate considerable acidity that can dissolve cave walls and speleothems. Possible microbially influenced corrosion include limestone corrosion residues composed of iron and manganese oxides and clays (eg. Lechuguilla and Spider Caves, New

Mexico; Northup & Lavoie, 2001; Northup et al. 2003), and sulfuric acid speleogenesis and cave enlargement (eg. Movile Cave, Romania, and Cueva de Villa Luz, Mexico; Vlasceanu et al. 2000).

Microbially induced mineralisation is documented in the formation of carbonates, moonmilk, silicates, clays, iron and manganese oxides, sulfur, and saltpeter. For example, sulfate generated by sulfur I sulfide-oxidising bacteria can be used as an electron-acceptor by sulfate reducers. This reaction produces bicarbonate that can complex with calcium, resulting in the precipitation of calcite in the form of subaqueous mantles (eg. Weebubbie Cave, Nullabor, Australia) (Contos et al. 2001). There is no clear idea as to the significance of biological involvement in speleothem formation, however, there are clues.

Studies of cave geomicrobiology are largely still qualitative in nature. Barton et al. (2001) and Jones (2001) offered critical guidelines for the biogenicity of 'objects' visualised in cave deposits: they must, be found in a liveable environment, show complex form, show representations by numerous specimens, be members of a multicomponent assemblage, show morphological variability, reproduction by biological means, exhibit a range of degradation, organic residues and exhibit biogenic isotopic features. Various microbiological techniques have

41 1.4 Geomicrobiology been used to illustrate that microbes are present in most spelean environments and commonly modify the composition of the fluids and/ or influence precipitation of various minerals, including calcite (e.g. Melim et al. 2001). Classical isolation combined with molecular phylogenetic techniques reveals the presence of microbial communities associated with speleothems (Caiiaveras et al. 1999). Enrichment experiments with microorganisms cultured from cave environments have aided in identifying dissolution and precipitation abilities of these cave microbes (eg. Groth et al. 1999a) and stable isotope techniques has provided information on the microbial contribution to processes of mineral formation (eg. Hose et al. 2000) and ecosystem bioenergenetics (eg. Sarbu et al. 1996).

Most bacteria in nature live as part of dynamic metabolically interactive assemblages, commonly referred to as biofilms, found covering most solid substrates (rocks, plants, man­ made structures) (Douglas & Douglas, 2000). The primary techniques for examination of biological material on mineral surfaces are transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy and environmental scanning microscopy

(ESEM) (Siering, 1998). Previous studies by Ray et al. (1997) and Douglas & Douglas, (2000) have shown the worth of ESEM for investigations of microbe-mineral relationships in natural microbial communities. Though SEM has been an important tool used to study cave microbial carbonates (Northup & Lavoie, 2001), ESEM allows the viewing of fully hydrated specimens that have not undergone structural or chemical alterations imposed by the extensive procedures necessary for viewing biological specimens in high vacuum necessary for conventional SEM.

Besides allowing visualisation of microorganisms in their natural form and as intact assemblages, ESEM also detects elements, especially those lighter that Si, which tend to be lost or masked by the processes used to prepare samples for conventional SEM (Douglas & Douglas,

2000).

The biogenicity of mineral-associated, purportedly biological features can be questionable and extremely difficult to resolve (Barton et al. 2001). Microbial activity has been directly or indirectly linked to the formation of many different minerals, however most geomicrobiology studies have focused their attention on the microbiological processes that are

42 1.4 Geomicrobiology associated with the development of carbonate deposits. Even with this focus, our knowledge of the microbial involvement in these processes has been limited by, i) the fact that there are few studies that have approached the issue from a geological perspective, ii) the fact that many geological studies of older deposits assume abiogenicity, iii) the fact that most geologists lack formal microbiological training, and iv) the scale of observation (Jones, 2001).

CaC03 speleothems predominate in most caves, and microbial studies have been conducted on stalactites, stalagmites, helictites, moonmilk, pool fingers and cave pearls.

Microorganisms have been found fossilised in carbonate speleothems (Jones & Motyka, 1987;

Polyak & Cokendolpher, 1992; Jones & Kahle, 1995; Melim et al. 2001). Fungi, algae and bacteria have all been implicated in the precipitation of carbonate dripstone in caves (Went, 1969;

Danielli & Edington, 1983). There is much evidence for rich and diverse chemoautotrophic and heterotrophic communities in caves (eg. Angert, et al. 1998; Sarbu, et al. 1996), it remains unclear however, what role, if any, these communities play in speleothem formation

1.4.2 Microbially Mediated CaC03 Precipitation

As some of the most abundant minerals on earth, carbonates are ubiquitous and highly reactive components of natural environments. Carbonate minerals play important parts in global carbon cycling, alkalinity generation, cycling of major and trace elements, and transfer of matter among oceans the continents and the atmosphere (Warren et al. 2001). Understanding carbonate precipitation has wide ranging implications from interpretation of biogeochemical

cycles, potential impact of increased atmospheric concentrations of C02 or reactive transport of radionuclides and trace metals in contaminated aquifers.

Bacterial precipitation of CaC03 has been reported in a variety of environments including hot springs, tidal mats and caves. It has been known since Boquet et al. (1973) that most

heterotrophic soil bacteria can induce CaC03 precipitation. Phillips & Self (1987) demonstrated that in soils with a high calcite concentration needle fibre-calcite formed within fungal mycelia and also encrusted rod-shaped microbes. Chafetz (1994) reported research carried out in the

43 1.4 Geomicrobiology field and laboratory conditions to demonstrate beyond doubt that CaC03 precipitation occurring within microbial mats was a process controlled by living bacteria and does not occur when the bacteria are dead, even in the presence of other living microorganisms.

Bacteria and fungi can precipitate CaC03 extracellularly through a variety of processes that include photosynthesis, ammonification, denitrification, sulfate reduction, and aerobic sulfide oxidation (Ehrlich, 1996; Castanier et al. 1999; Riding, 2000). Castanier et al. (1999)

proposed biologically mediated or active precipitation of CaC03 where carbonate particles are produced by ionic exchanges through the cell membrane of heterotrophic bacteria in an environment enriched in organic matter. Initially, this involves the adsorption of Ca2+ and

Mg2+ ions to negatively charged cell surfaces and the cell then acts as a nucleation site.

Subsequent CaC03 precipitation may be active or purely inorganic. Riding (2000) noted that microbial production of extracellular polymeric substances (EPS), which trap sediments, is often critical to the creation of microbial carbonates. Terrestrial oncoids (microbially formed carbonate constructions from dolestones, Cayman Islands) developed when calcifying filaments and spores trapped and bound detritus within the associated mucus (Jones, 1991). These resemble cave pearls, a speleothem that has been suggested to have a microbial association during formation (Gradzinski, 1997).

More recent studies have attempted to identify the factors that control the contribution of microorganisms to carbonate precipitation. Further progress in this field has been made in non-cave environments. Van Lith et al. (2003) found that only pure cultures of metabolising sulphate-reducing bacteria, isolated from hypersaline lagoons in Brazil, induced calcium­ dolomite and high magnesium-calcite precipitates indicating that the carbonate nucleation takes place in the locally changed microenvironment around the bacterial cells. Dittrich et al. (2004) showed that picocyanobacteria were involved in fast and effective calcite precipitation in an oligotrophic lake. Whether by saturation or nucleation they observed small calcite crystals produced by eukaryotic picoplankton whereas cyanobacterial picoplankton produced micritic carbonate indicating that different cells may induce very different, distinct precipitation processes.

44 1.4 Geomicrobiology As previously discussed, geological formations in caves (speleothems) like stalagmites and stalactites, are mineral depositions formed by precipitation of carbonates from ground water. Extensive documentation of microbial precipitation of CaC03 exists in non-cave literature, biogenic carbonates in particular have been studied since the late 19th century (eg. stromatolites; Chafetz & Buczynski, 1992). Microorganisms are believed to affect carbonate precipitation both through affecting local geochemical conditions and by serving as potential nucleation sites for mineral formation (McGenity & Sellwood, 1999). In natural environments, the primary means by which microorganisms promote CaC03 precipitation is by metabolic processes that increase alkalinity (Fujita et al. 2000). However, investigators have not established whether cave carbonate material has a similar origin.

Some of the most intriguing work on cave fungi associated with speleothem formation was conducted by Went (1969). The author made the interesting discovery that the growth of stalactites in Lehman Caves, Eastern Nevada, was associated with a fungus, Cephalosporium lainellaecola. This discovery was made using a special microscope mounted horizontally on an adjustable bracket sliding along a vertical steel bar so that stalactites could be observed in situ within the cave. He found that fungal hyphae occurred in a drop of water at the end of a straw stalactite and that strings of tiny calcite crystals tended to form along them. The hyphae not only functioned as crystallisation nuclei but also prevented the crystals from being removed with the falling drops. Perhaps actinomycete filaments act in the same way. Actinomycetes isolated from either dripping waters or rock in Altamira Cave showed the ability to produce crystals and therefore could play a role in the deposition of CaC03 polymorphs on the rock surface (Laiz et al.

1999). Although there is no known role for CaC03 in bacterial metabolism, certain organisms precipitate calcite during their growth (Buczynski & Chafetz, 1990). Groth et al. (2001), found that 45% of isolates from stalagmites in Grotta dei Cervi, Porto Badisco, Italy were able to precipitate CaC03 in culture medium. Organisms such as Achromatium oxaliferum contain internal calcite inclusions during growth (Head et al, 1996). There is also an established role for bacteria in the nucleation of CaC03 precipitation for stromatolite formation (Ehrlich 1999; Laval et al. 2000).

45 1.4 Geomicrobiology Various experiments have shown that bacteria may be replaced or encrusted by inorganic materials resulting in the fossil preservation of bacterial morphology. There are reports of bacteria preserved in carbonate rocks (Folk 1993; Jones 1995; Trewin & Knoll 1999) and a few reports of iron-oxidising bacteria preserved in carbonate speleothems in caves (Polyak

& Cokendolpher, 1992). However, although microfossils have been identified in carbonate speleothems, no direct connection with active precipitation processes in the formation of these features has been demonstrated. Bacterially induced changes in solution chemistry can be a passive process (eg. stromatolites; Chafetz & Buczynski, 1992). Evidence that microbes play a role in the formation of cave carbonates is still largely circumstantial and based on their physical presence. The question still remains whether the organisms identified are actively involved in speleothem formation, or simply buried during mineral precipitation (Polyak & Cokendolpher,

1992). Of special interest is the speleothem moonmilk. As discussed in Section 1.2.2, the wet pasty forms of moonmilk are so striking that some special explanation for their origin seems to be necessary. Caii.averas et al. (1999) suggested that bacteria present in caves may play a role in the formation of moonmilk deposits as microbial communities predominantly composed of different species of the genus Streptomyces were found in association with hydromagnesite and needle-fiber aragonite deposits in Altamira Cave.

1.4.3 Moonmilk

Moonmilk is a widely distributed, secondary formation and refers to the very hydrated white spongy /pasty or powdery masses found coating walls and speleothems in caves. It is not a mineral, it is a speleothem. It is often described as having a cottage cheese-like consistency and may be composed of several carbonate minerals. The historical term Mondmilch (=calcite moonmilk) is related to the proper type locality, the cave Mondmilchloch from South-Pilatus near Lucerne, Switzerland. Mondmilch was first mentioned by Agricola (1546, p. 194) and described by Gesner (1555) after visiting the cave Mondmilchloch (Fischer, 1988). This was without consideration of the actual mineral composition of the deposits. Moonmilk became well

46 1.4 Geomicrobiology known throughout Europe and used as a medication (Scheuchzer, 1752; in Fischer, 1988). In the

1 16 h and 11'1" centuries, physicians in Europe used dried moonmilk from caves as a dressing for wounds. Apparently moonmilk would stop the bleeding and act as a dehydrating agent.

However, some also believed it had curative properties. It is rather interesting that modem research or theories have discovered bacteria associated with moonmilk include actinomycetes that, as previously discussed, possess antibiotic properties.

Many descriptions of moonmilk are not only related to calcite growth, although the original term mondmilch from the cave Mondmilchloch refers to calcite precipitation and does not represent the phenomenon for speleothems in general. Hill & Forti (1986) suggested that texture rather than composition is implied by the term 'mondmilch'. Fischer (1988) defined mondmilch as a calcite microcrystalline or needle-crystalline speleothem with a minimum calcite content of 90 % weight, for the purpose of distinguishing true calcite mondmilch from other carbonate speleothems (< 90 % weight calcite) and other subterranean deposits, (eg. ferromanganese, sulfates, phosphates, silicates). The mineralogy and crystallography of potential mondmilch samples can be easily proved using X-Ray Diffraction Analysis (XRD) and scanning electron microscopy (SEM) methods (Fischer, 1988). Numerous synonyms in different languages exist for mondmilch, including the English version moonmilk.

True calcite moonmilk (mondmilch) has been found in many caves all over the world and appears to be particularly abundant in caves of cool temperature and high humidity. In warmer semi-arid regions limestones contain significant amounts of magnesium and moonmilk deposits may consist of a number of magnesium minerals including hydromagnesite, magnesite, huntite or dolomite (Moore & Nicholas, 1964). In Australian caves moonmilk has been documented in a variety of forms including, thin dry wall coatings, white cheese-like pasty forms at the bottom of rimstone pools or wall niche deposits, stalactites, cauliflower-like deposits and fluffy fungus-like forms in Jenolan Cave, NSW (http:/ /www.speleonics.com.au; maintained by J. Rowling). The origin of moonmilk deposits is highly contested among the literature. Hill & Forti (1986) cite four main theories as to the origin of moonmilk: i) freezing in ice caves, ii) precipitation from groundwater in which there is an agent which prevents the

47 1.4 Geomicrobiology crystals from growing large, (the theory preferred by Hill & Forti), iii) a disintegration product of bedrock, and iv) a by-product of the life cycle of various microorganisms. Each of these theories taken individually has its pros and cons as an explanation for the development of moonmilk.

White (1976) suggested that moonmilk in alpine caves may be precipitated chemically at low temperatures as hydrated carbonates, however these forms are only stable at low temperatures. Also freezing in alpine icy caves does not explain the occurrence of temperate and tropical moonmilk. Rapid precipitation of substances will generally result in small crystal size

and this occurs near cave entrances where precipitation is due to both outgassing C02 and evaporation of water (http:/ /www.speleonics.com.au; maintained by J. Rowling). It also occurs where gypsum is being produced. However, this does not explain the pasty textured moonmilk or that speleothems in cave entrances are hard and crystalline and quite unlike moonmilk.

Moonmilk is usually considered a depositional product. However, Hill & Forti (1986) suggested it can also form by corrosion processes. It is suggested that moonmilk could be a product of microbial metabolism which could biochemically corrode underlying bedrock. There are of course various types of bedrock and numerous disintegration processes that can occur within caves and though calcite deposits may be formed, they do not have the pasty texture that moonmilk is famous for. One common cause of bedrock disintegration is bat guano

(http:/ /www.speleonics.com.au; maintained by J. Rowling). It has been suggested that the thin films of moonmilk may be a result of bat guano bedrock disintegration, however this would not explain the moonmilk films in Entrance Cave, Tasmania, Australia, as there are no bats inhabiting Tasmanian caves due to cool air temperatures. Gradzinski et al. (1997) concluded that moonmilk deposits from several caves in Poland might be the result of microbial degradation of the host rock, as well as, or in place of microbially mediated precipitation of calcite. Conversely, in Spider Cave, New Mexico, SEM pictures of the microcrystals that make up moonmilk do not show the evidence of weathering that would be expected if microbial corrosion routinely resulted in moonmilk production (Northup & Lavoie, 2001).

48 1.4 Geomicrobiology It is generally accepted that moonmilk might be the by-product of the life-cycle of various microbes, although the question remains whether the organisms identified are actively involved or simply buried during mineral precipitation. A number of organisms have been

isolated from cave environments and shown to precipitate CaC03 in the laboratory (Danielli &

Edington, 1983; Groth et al. 2001). One very interesting point, a thick wall-niche deposit of moonmilk in Jenolan Cave is recorded as having been damaged by a person's handprint to at least 1 cm depth, yet 8 years later the print is/was no longer visible

(http:/ /www.speleonics.com.au; maintained by J. Rowling). Given the long length of time for usual speleothem growth (1 inch in 100-150 years; Section 1.2.2) this prompts the question as to whether the moonmilk was "alive" or not. Putative cells and an organic matrix can be frequently seen in moonmilk samples with SEM or in thin sections, but not in all cases. A wide range of microbes, particularly bacteria, streptomycetes but also fungi, algae and protozoa, can be cultured from moonmilk often in high densities (Northup et al. 2000).

Williams (1959) inoculated moonmilk samples from several caves in South Wales into various nutritional media and isolated eight species of heterotrophic bacteria belonging to the genera Bacillus, Micrococcus, Bacterium, and Streptomyces. In one culture a Gram-negative rod, a thiobacteria, was detected with the ability to produce CaC03 in crystalline forms similar to those found in moonmilk. Danielli & Edington (1983) isolated a wide range of colony types (mostly

Gram-negative cells) from moonmilk collected from caves in Wales and calcite precipitation was a common factor of these isolates. These authors suggested that cells were using the organic salt anion for energy and dumping the calcium as a waste product. When the calcium exceeded the solubility threshold, precipitation resulted. CaC03 encrusted cells then served as a nucleation site for further crystal formation. Gradzinski et al. (1997) proposed stages in the progressive formation of moonmilk where cells and an organic matrix first provide a structural framework; then, active bacterial cells are calcified and the extracellular organic matrix fills the remaining space with calcite. Although there is no known benefit of CaC03 precipitation in bacterial metabolism, detoxification of calcium has been suggested (Northup & Lavoie, 2001).

49 1.4 Geomicrobiology A very interesting cave phenomena usually associated with moonmilk deposits is the white/ grey silvered films which are covered in reflective dots and usually occur on walls and ceilings within the Twilight to Dark Zones of limestone caves and lava tube caves Gones, 1995)

Although there are no published works that this author could find, it seems to be widely accepted that the white/ grey coating is colonies of actinomycetes. The surface of these colonies is hydrophobic but the filamentous structures are hydrophilic and the droplets are attached to the end of these filaments. Lake & Rowling (pers. comm. J. Rowling, Aristocrat Technologies

Australia, 2004) collected some liquid from colonies at Jenolan Caves and investigated the mineral aspects of the liquid, and found that it was almost entirely calcite. These investigators postulated that perhaps these actinomycetes contributed to moonmilk deposits. The ability to form CaC03 polymorphs seems widely distributed among environmental actinomycetes; 19 out of 31 cave strains isolated and tested by Laiz et al. (1999) produced a considerable amounts of crystals in both solid and liquid media.

Microbial precipitation does not explain all forms of moonmilk. It is likely that there may be abiotic forms as well. An extensive survey of moonmilk deposits from high-altitude caves in the Italian Alps revealed no evidence of microbial involvement in calcite precipitation.

A review of factors contributing to the formation of moonmilk deposits in these alpine caves includes elevation and temperature, along with surface cover of soils and conifer forests and with low discharge rates of seepage water and high humidity (Borsato et al. 2000). However, the majority of samples were from fossil deposits (Borsato et al. 2000). Given the wide variability of minerals that may form moomilk it is not surprising that several mechanisms, biotic and abiotic, have been proposed for its formation, one or more of which may be involved in the deposition of moonmilk in a particular form or particular type of cave.

Biotic and abiotic hypotheses for the formation of moonmilk do not need to be mutually exclusive (Northup & Lavoie, 2001). Given the variety of mineral types involved and the range of physicochemical conditions, microbes are clearly involved in the formation of moonmilk by dissolution or by serving as nucleation sites in some cases, but they may play a minor or negligible role in other cases. Friedman & Sanders (1978) noted that "Purely inorganic chemical

50 1.4 Geomicrobiology reactions can take place only where simple organisms are totally absent. At the surface of the earth, environments devoid of such organisms are uncommon." That same observation is true for subsurface environments. Studies of dissolution and precipitation of carbonates, moonmilk, silicates, clays, iron and manganese oxides, sulfur, and saltpetre in caves span only a few decades. A variety of organisms with biogenic potential have been discovered and some fascinating systems and environments have been described from caves. These studies provide insights into biomineralisation in general, and in the formation of speleothems in particular

(Northup & Lavoie, 2001).

51 1.5 Significance

1.5 Significance

1.5.1 Biodiversity and Conservation Value

Biodiversity is the variety of all life forms: the different plants, animals and microorganisms, their genes and the ecosystems to which they belong. Australia is one of the most biologically diverse countries in the world with a large portion of its species found nowhere else in the world (1/5 of the world's diversity). Biodiversity underpins the processes that make life possible. Healthy ecosystems are necessary for maintaining and regulating atmospheric quality, climate, fresh water, marine productivity, soil formation, cycling of nutrients and waste disposal. Thus we depend on biodiversity for our survival and quality of life.

At the 1992 Earth Summit in Rio de Janeiro, world leaders agreed on a comprehensive strategy for "sustainable development", meeting our needs while ensuring that we leave a healthy and viable world for future generations (Department of Environment and Heritage,

Australian Biological Resources Study website; http:/ /www.deh.gov.au/biodiversity I abrs/).

One of the key agreements adopted at Rio was the Convention on Biological Diversity setting commitments to sustainable development. The two main goals established by the Convention were the conservation of biodiversity and the sustainable use of its components. The most significant impediment to the conservation and management of biodiversity is our lack of knowledge of it and the effects of human population and activities on it. Accordingly, a taxonomic perspective is necessary to conserve biodiversity and achieve sustainable development.

A taxonomic perspective includes providing underlying taxonomic knowledge of biodiversity and the environmental factors influencing species distribution in microhabitats.

Providing baseline information on the composition and distribution of cave microbial communities is essential to aid the conservation of cave microbial communities from human impacts.

52 1.5 Significance 1.5.2 Bioprospecting

A critical element in drug discovery based on microbial extracts is the isolation of

unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Together with their importance in soil ecology, actinomycetes are best known as a source of antibiotics. This became apparent in 1940, following Selman Waksman's seminal discovery of acti.nomycin (Waksman & Woodruff, 1940) and was fully realised by the 1980s when acti.nomycetes accounted for almost 70% of the world's naturally occurring antibiotics

(Okami & Hotta, 1988). Acti.nomycetes, represent an important source of biologically active compounds whose members have unparalleled ability to produce diverse secondary metabolites. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets' (Donadio et al. 2002). Thus acti.nomycetes are a group of high economic, social and health significance.

In the past two decades there has been a decline in the discovery of new lead compounds from common soil-derived actinomycetes as culture extracts yield unacceptably high numbers of previously described metabolites (Mincer et al. 2002). Natural products continue to be a potent source of novel drugs and other bioactive compounds despite the

I emergence of combinatorial chemistry. The important attributes of natural products are their molecular diversity, still very much greater than that of combinatorial libraries, and their biological functionality (Nisbet & Moore, 1997). For this reason cultivation of rare or novel actinomycete taxa has become a major focus in the search for the next generation of pharmaceutical agents (Bull et al. 2000). The pharmaceutical industry has a strong interest in the acquisition of novel acti.nomycete biodiversity in the search for new lead compounds. There is strong incentive therefore to discover novel microbes whether it is done by exploiting molecular biology and/ or by exploring unusual biotopes (Colquhuon et al. 2000). Due to this interest significant biodiversity has been targeted and described from accessible environments. Williams et al. (1993) stated that one approach to the isolation of novel acti.nomycetes is to concentrate on understudied environments or substrates while using appropriate selective isolation techniques or to investigate habitats in which one or more of the environmental factors (eg. temperature,

53 1.5 Significance pH, aeration, or osmotic stress) are extreme. This has lead to the strategic targeting of extreme or unusual ecosystems. The importance of this area of rest:;arch has been recognised by the international research community, for example by recent EU funding of a new initiative at the

University of Newcastle upon Tyne ("New Approaches to the Discovery of Novel Bioactive

Compounds from Natural Actinomycete Communities").

Caves are unique ecosystems exposed to extreme environmental stresses. The limiting environmental characteristics of caves, little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate, provide ecological niches for highly specialised and very diverse microbiota. Preliminary investigations of microbes isolated from the most remote and least human-impacted regions of Lechuguilla Cave, New Mexico, have highlighted their potential as sources of anti-cancer treatments, because of their ability to kill breast cancer cells (Northup & Mallory, 1998). Novel actinomycetes isolated from caves represent an important, potentially valuable biotechnological resource for the screening and discovery of novel bioactive compounds due to their origin from a unique and as yet poorly studied environment.

1.5.3 Bioremediation

Microbial biodiversity is a reservoir of resources that remains relatively untapped.

Microbes are the only life-forms that have been encountered in the deeper regions of the earths crust. Subsurface microbes with novel metabolic properties may be of potential value to industry for applications in bioremediation and biotechnology (eg. Gold, 1992; Boone et al. 1995;

Stevens & McKinley 1995; Bale et al. 1997; Krumholz et al. 1997, 1999; Chandler et al. 1998;

Whitman et al. 1998; Kieft et al. 1999; Takai & Horikoshi, 1999). In spite of recent findings, many of these microbial habitats remain poorly characterised mainly due to difficulties associated with access and sampling. Caves provide an accessible point of entry to the shallow subsurface.

Throughout.the world, organic and inorganic substances leach into the subsurface as a result of human activities and accidents, for example agricultural pesticides, landfill leachate.

54 1.5 Significance There, the chemicals pose direct or indirect threats to the environment and to increasingly scarce drinking water resources. At many contaminated sites the subsurface is able to attenuate pollutants that, potentially, lowers the costs of remediation. Natural attenuation comprises a wide range of processes of which the principle mediators are the microbiological component, which is responsible for intrinsic bioremediation, and can decrease the mass and toxicity of the contaminants by transforming or mineralising pollutants and is, therefore the most important

(Christensen et al. 2001; Roling & van Verseveld, 2002). Of particular relevance is the ability of subsurface microbes to induce formation of CaC03 minerals which presents an opportunity to develop and in situ bioremediation techniques for groundwater contaminated with divalent metals or radionuclides (Fujita et al. 2000). Reliance on intrinsic bioremediation requires methods to monitor the process. Knowledge of the subsurface geology and hydrology, microbial ecology and degradation processes can be used to monitor the potential and capacity for intrinsic bioremediation in the subsurface.

1.5.4 Biodeterioration & Biomzneralisation Processes

1.5.4.1 Palaeolithic Frescoes and Rock Art in Hypogean Environments

It is now well recognised that wall paintings can be severely damaged by microbial growth (Ciferri, 1999). It has been reported in the literature that pigment formation, crystal growth and other types of biodeterioration processes related to microbial activity affect rock paintings and frescoes in cave environments. In studies on the bacterial community associated with such deterioration, members of the actinomycetes both previously cultured and novel, are frequently cultivated (Sorlini et al. 1987; Weirich, 1989; Petushkova et al. 1990; Altenburger et al.

1996, 2002; Rolleke et al. 1996; Groth et al. 1999a; Wieser et al. 1999; Gurtner et al. 2000; Heyrman

& Swings, 2001; Gurtner et al. 2001; Heyrman et al. 2002). Studies in Altamira and Tito Bustillo

Caves, Spain, demonstrate that rock art paintings are coated by dense networks of bacteria, mainly actinomycetes. Identified damage includes: i) covering (scattered coloured spots, whitish powdery patinas, staining) of paintings by the microbial communities themselves and/ or by

55 1.5 Significance their metabolic activity (including biofilms and bio-induced precipitates); ii) chemical alteration, such as microbial mediated dissolution; and iii) mechanical alteration, such as rock substrate breakdown.

Bacteria can use organic compounds from the paint layer as growth substrates, producing acids, which cause discolouration of the paint or changes in its consistency. For example, iron-enriched pigments in rock art act as a substrate for attachment and a mineral supply for growth. In favourable conditions the bacteria present can change the colour of the paintings from the reddish yellow hues characteristic of iron pigments to a dark yellowish colour as a result of microbial metabolism. As noted previously in Section 1.4, some cave bacteria may play an important role in the precipitation and/ or deposition of CaC03 speleothems. Many of the actinomycetes isolated from caves are able to precipitate CaC03 crystals. These bacteria can induce constructive (calcification, crystalline precipitates) and destructive (irregular etching, spiky calcite) fabrics on the paintings and/ or surrounding rock.

Microbes can penetrate into the painting and its bedrock resulting in mechanical destruction of the cultural heritage (dissolution, etching of the host rock). In a study by Ca:fi.averas et al. (1999) a

Streptomyces xanthophaeus strain isolated from Tito Bustillo Cave walls was inoculated onto stalactite slices which showed pitting formation after only three months of culture in the laboratory illustrating a bacterially mediated calcite dissolution process. Because of this ability it has been proposed that these bacteria and others are directly or indirectly involved in constructive and destructive biomineralisation processes in caves (Laiz et al. 1999).

1.5.4.2 Monuments

Interestingly, a group of geomicrobiologists in Spain are following a unique view of biomineralisation processes by suggesting using bacterially induced carbonate mineralisation as a novel and environmentally friendly strategy for conservation of ornamental stone monuments.

Increasing environmental pollution in urban areas has been endangering the survival of

56 1.5 Significance carbonate stones in monuments and statuary for many decades. Numerous conservation

treahnents have been applied for the protection and consolidation of these works of art. Most of

them, however, either release dangerous gases during curing or show very little efficacy. There have been a number of studies looking at biomineralisation processes, particularly bio-mediated

calcite precipitation, for monumental stone conservation (Di Bonaventura et al. 1999; Tiano et al.

1999; Urzi et al. 1999), for example, Myxococcus xanthus -induced CaC03 precipitation efficiently

protects and consolidates porous ornamental limestone. (Rodriguez-Navarro et al. 2003). Calcite­

precipitating cave isolates have the potential to contribute in this area.

1.5.5 Management Issues

Cave environments are generally quite stable. Diurnal changes have little effect on the

cave microclimate. Similarly, seasonal variations in temperature and humidity are relatively

minor. Air movement is regulated largely by cave morphology and if present, by the active watercourse. There are low numbers of macroscopic living organisms in caves, mostly insects

and spiders. In such a stable environment, microbial growth is the main threat to the preservation of the cave environment. The effects of microbial growth are exacerbated by human impact both on the external cave environment (eg. pollution, changed land use) and by visiting

the caves. Visitation produces a more direct and pronounced effect. Visitors produce variations in environmental conditions and increase microbial dispersal and colonisation, Humans can introduce foreign organisms from the surface environment that can establish in caves and they leave behind organic material (lint, hair, skin flakes etc) that provide a rich nutrient source for the proliferation of micro-organisms.

There are implications for Heritage Management in the case of hypogean environments containing Palaeolithic rock art. Pigment formation, crystal growth and other types of biodeterioration processes related to microbial activity affect rock paintings and frescoes in cave environments. These bacteria induce constructive effects such as calcification, crystalline

57 1.5 Significance precipitates, covering) and/ or destructive fabrics such as irregular etching, spiky calcite, substrate break-down and dissolution.

There are also implications for cave management issues include the impacts of changes in hydrology, cave sediment contamination on speleothems, and tourist cave lighting upon the natural microbial communities existing within cave microhabitats. Whether microbial communities are actively or passively involved in speleothem formation, disruptions to the natural communities will have an effect on the health and continued formation of speleothems and cave systems.

58 1.6 Conclusion

1.6 Conclusion

Cave environments represent one of few remaining isolated planetary habitats, in terms of human impact and the characterisation of novel microbial diversity. In the past, the study of microbial communities and biogeochemical processes in hypogean environments is mainly related to the fact that microbes affect cultural heritage properties that humans wish to protect and we owe much of our initial knowledge of cave microbiota to these studies. These studies may not necessarily reflect the biodiversity in 'natural' cave systems ie. those that are not heavily impacted by tourism. Compounding this, culture-based studies often have no 16S rRNA gene sequence data for isolates. Most published studies use morphological and biochemical means of identification, rather than phylogeny, to characterise cave strains to the genus level only. Thus it is difficult to make detailed comparisons at the species level between cave environments. Sequence data is available for described novel species from caves, however, little has been published about the cave environments that these novel species were isolated from.

It is widely accepted that only - 1 % of microbes are cultured in the laboratory. Culture­ independent methods are being increasingly used.to describe the composition of microbial communities and reveal significantly broader diversity than culture-based studies. Nevertheless, to date our knowledge of bacterial communities in caves is largely due to culture-based studies.

The past decade has seen a rapid increase in published investigations of microbial ecology in caves. However, the diverse range of types of caves (Eg. sulfur caves, carbonate caves, aquatic caves, tourist/show caves, restricted access caves) and microhabitats (Eg. acidic biofilms on walls, filamentous microbial mats in sulfur waters, aquatic microbial mantles, Palaeolithic rock art, cave walls, ferromanganese deposits, sediments) studied and the geographic separation of sites (Romania, Italy, Australia, Mexico, Spain, North America) makes it difficult to draw many comparisons or conclusions about cave microbial diversity (Eg. Sarbu et al. 1996; Angert et al.

1998; Vlasceanu et al. 2000; Holmes et al. 2001; Summers-Engel et al. 2001; Schabereiter-Gurtner et al. 2002, 2004; Northup et al. 2003; Chelius & Moore, 2004; Barton et al. 2004). Despite this recent expansion of our knowledge, literature on cave microbial communities, their distribution and

59 1.6 Conclusion taxonomic diversity, is limited and restricted to only a few caves world-wide, predominately in the northern hemisphere. In the southern hemisphere investigations of microbial diversity in caves is represented by only one publication. Holmes et al. (2001) investigated microbial diversity in unusual aquatic formations, mantles of mucus and biological material associated with crystalline material in submerged passages in the Nullabor Caves, Australia; a very unique microhabitat thus most likely not representative of general cave microbial biodiversity in the southern hemisphere. Molecular techniques are only recently being applied to geomicrobiological questions in hypogean environments (Eg. ferromanganese residues in

Lechuguilla Cave; Northup et al. 2003), and as yet there are no published culture-dependent reports of microbial communities associated with moonmilk deposits.

The description of the composition of microbial communities is an important starting point in studies of microbial biodiversity and sets the stage for fundamental studies concerning how these populations function (Morris et al. 2002). The microbial diversity of as yet poorly studied environments is being increasingly explored by molecular detection methods (eg.

Eppard et al. 1996; Rheims et al. 1996, 1998; Sarbu et al. 1996; Vlasceanu et al. 2000; Holmes et al.

2001; Summers-Engel et al. 2001; Schabereiter-Gurtner et al. 2002, 2004; Northup et al. 2003).

While molecular methods are valuable tools in characterising the microflora, isolation and culturing are still required for describing the microbial diversity, especially in the case of novel taxa (Palleroni, 1997).

60 Chapter 1: Introduction

SECTION 2:

MICROBIAL BIODIVERSITY IN TASMANIAN CAVES

Chapter 1: Introduction

Some of the deepest, longest and most beautiful caves in Australia are found in

Tasmania. Tasmanian caves are of mixed character (wet/muddy vs. dry) and range from commercially used caves to new or unexplored caves. Due to our southerly latitude, the caves in

Tasmania are colder and wetter than elsewhere in Australia with temperatures ranging as low as 4-7 °C. An interesting point to note is that there are no bats in Tasmanian caves, probably due to the cool air temperature in these caves. The Ida Bay Karst area is located in southern

Tasmania, mostly within the Tasmanian Wilderness World Heritage Area {Figure 1.1). Most of the karst retains native vegetation cover, which is wet sclerophyll forest and rainforest. The Ida

Bay Karst developed in Ordovician Gordon limestone from 510 to 439 million years ago (Mya), outcropping between 50 and 300 m above sea level. Cave development is substantial, with more than 140 cave entrances and in excess of 20 km of mapped passage, and predominantly CaC03 speleothems (Eberhard, 1999). The extensive cave systems in this region have a long and complex history of development, with Cainozoic (65 MYA to present) cold climate change exerting a major influence (Goede, 1968; Kiernan, 1982). Environmental conditions within the cave systems are thought to have changed little since they formed, except for periodic glacial sediment inclusion.

61 ....

Legend

Oual«n#y • Sikeout Sl'diment. OJallmlfY • Cek•eous Se.Ja'9maf"l·Glaci.al ~P~ FNt!Sts D Ou&t@mary. Other l,h:onsolldat!d Sedm«its

T«W-/·Sll~Sedirnenb Tet1art • Calc.eous Sediments Tertary·8as.al1 Crf:taceout • "'kallne lnt"usions

- Triffsit...kns5k-Basalt Trtasstc - CMbOnae:KttS Sedments C:J Pl"fmian-Triassk:·SiliceousSedment

- SUo-()r(orUi • Sii~ 5edm«lts - ~Oevcrian · Slkerus Sediments

- cambflen • Basalt m c.mo""'-"""" CWnliflan - SilkeousStdlrMnt'S CJ c.tnbNn • Felsic Volclrics [:::J Cam«!on-,,_ V_, c::J Cembrian- Ore Oeposits. !Ulran"'6G-M<1kcoml)le:•es1 Q c.ntirian - OreOtposits t Basall-gf*'fl'a<.ke~s l Neoprotero.r.oic· Basell mr1-"""'""·""""" Neopl"otemioic - Sedment

Figure 1.1: Map of Tasmania depicting the World Heritage Area and Ida Bay karst region. Entrance-Exit Cave system and Loons Cave are located in the Ida Bay karst. Overlay detailing geology of Tasmania, including calcerous sediments of the Ida Bay karst region.

Data provided by National Parks and Wildlife Service Tasmania.

ou.wn..;- ewtS~

~- Clllc•.aus~ o~- iO.t#llll P"91a11fHUff CJ"'-"">·""*'-"'--­ lfftary- Sllcw.n ~

T~-Catc..wt~

r...., . ~,,,,.

0-elaCAOul - Nkllllne "*usionl

..WM - ~ - T~ff• • BH"'1

~ . c.banac9CU5 s.canents Q Plfmian-Tt1Mlic . Siiiceous s.dnteots c::J P«miM'I • Calc•eout S9dlmtnb OtYonlan . Ol'...W. - SUo-~ - Sllcews S9dlrMntl _.'aliai.-ili' llil>W~ ~ 'Ht!ritage Area - ~-"""""'C.ntirian -Sltbo1ot1~ .

Ida Bay karst: Entrance-Exit Cave Loons Cave

62 Chapter 1: Introduction

Figure 1.1: Map of Tasmania depicting the World Heritage Area and Ida Bay karst region. Entrance-Exit Cave system and Loons Cave are located in the Ida Bay karst. Overlay detailing geology of Tasmania, including calcerous sediments of the Ida Bay karst region.

Data provided by National Parks and Wildlife Service Tasmania.

Ida Bay karst: Entrance-Exit Cave Loons Cave

62 Chapter 1: Introduction

The biological importance of the Ida Bay caves has been recognised for more than 100

years, beginning with an article published in Scientific American describing the spectacular glow

worm display in Entrance Cave (Anon. 1895 in Eberhard, 1999). Over the years, many rare and

endemic obligate cave fauna have been discovered and described from Ida Bay caves resulting

in this region being widely recognised as containing one of the more diverse and significant

assemblages of cave fauna in Australia's temperate zone (Richards & Oilier, 1976).

The Entrance-Exit Cave System is a site of high biological significance, the most

outstanding biological feature of the caves being the glow worm display. The Entrance Cave

subsystem, in particular, is the type locality for many obligate cave dwelling fauna (Richards &

Oilier, 1976). Near the entrance and extending for some distance into Entrance Cave there is a

very significant cave fill deposit consisting of a conglomerate of rounded boulders and pebbles

set in a fine matrix and thoroughly indurated. This deposit has not been studied in detail but an

intelligent guess is that it is sediment of glacial times, when solifluction was prevalent but

running water was much reduced (Richards & Oilier, 1976). The significant feature is that this

, deposit extends to roof level which possibly means it blocked the cave completely at one stage

allowing 'evolution' in isolation and has since been largely removed by subsequent stream

action (Richards & Oilier, 1976).

Loons Cave, although very dose in proximity, is a very different system to the Entrance­

Exit Cave System. Loons Cave essentially consists of a single, narrow, low energy stream

passage that appears to be fed primarily by waters of seepage origin not a streamway

originating from the surface (Household & Spate, 1990). The cave is reasonably well decorated

with speleothems that are generally massive and robust. Loons Cave is commonly used as an

"outdoor experience" locality for school and recreational groups and is therefore a site of high

human impact in contrast to the majority of the Entrance - Exit Cave system.

Deposits of moonmilk are a common feature of many Tasmanian caves (Goede, 1988).

Despite their abundance they are amongst the least studied and understood of any of the cave

63 Chapter 1: Introduction

deposits. Very large moonmilk deposits are evident in Exit Cave occurring as a uniform or

botryoidal layer that covers stalactites, cave walls, ceilings and floors. Entrance Cave is also

known to have moonmilk deposits although on a much smaller scale than Exit Cave. Within the

Entrance Chamber of Entrance Cave, just beyond the cave mouth, large white mats with silvered

droplets (similar to those described in Section 1.4.3) are visible on the ceiling rock. These have been anecdotally described as being actinomycete colonies by enthusiastic cavers, though this has previously not been investigated. The white mats are visible past the Twilight Zone of the

cave and in the Dark Zone, however not to the same extent. Although there are no moonmilk

speleothems in Entrance Cave per se, it was discovered during the course of this study that there

are large deposits of moonmilk beneath the sediment throughout the cave.

The focus of this research was the characterisation of microbial biodiversity from

Tasmanian caves (Entrance-Exit Cave system and Loons Cave) in 3 microhabitats; sediments, speleothems and moonmilk deposits. Isolation of pure cultures reveals only a minor fraction (-

1 %) of the actual biodiversity in an environment. Culture-independent 16S rRNA gene sequence analyses have opened the way to study bacterial communities in environmental samples without prior cultivation and reveal a significantly broader diversity than culture-based studies

(Amann et al. 1995; Head et al. 1998; Hugenholtz et al. 1998). Bacterial diversity in Tasmanian caves have not been investigated using culture-independent techniques and to date there is no published culture-independent study on moonmilk worldwide. Thus classical isolation and molecular detection methods (DGGE, 16S rRNA gene clone library analysis) were used to compare culturable vs. non-culturable biodiversity, particularly of the actinomycetes who appear to dominate isolations from culture-based studies of heterotrophic cave systems. To expand our knowledge of cave microbial diversity, phylogenetic analysis was used to determine diversity at the species level and to infer ecological function where possible. The biodiversity described acts as a baseline for assessing environmental impacts and also identifying factors influencing microbial diversity.

64 Chapter 2: Materials and Methods

Chapter 2: Materials and Methods

2.1 Site description and sample collection

2.1.1 Entrance-Exit Cave System

The Entrance Cave subsystem has a simple cave opening where Mystery Creek goes underground and is located approximately 2 km from Ida Bay, along the South Lune Road. The cave follows the course of Mystery Creek entering the north side of Marble Hill, also known as

Caves Hill, at an elevation of 115 m (Richards and Oilier, 1976). The cave floor is a riverbed, covered with large boulders and cobbles. Water and nutrients are contributed to the lower level passages by the active inflow stream Mystery Creek, whereas the upper level passages are dry.

Mystery Creek re-emerges via a non-negotiable route into Exit Cave and a subterranean section of the D'Entrecasteaux River draining out of the south side of Marble Hill. Exit Cave is the longest cave in Australia with greater than 15 km of passages, generally large sized. Mystery

Creek is also an important inflow stream to the Exit Cave subsystem, contributing water and nutrients. There are also many smaller feeder passages in the Exit Cave subsystem with low energy streams and more than 100 other known caves in the Ida Bay karst that are predominantly vertical shaft caves on the slopes of Marble Hill and connect with the Exit Cave subsystem at depth.

2.1.2 Loons Cave

Loons Cave essentially consists of a single, narrow, low energy stream passage that appears to be fed primarily by waters of seepage origin (Household & Spate, 1990). The natural, undisturbed substrate in this stream consists of a lightly cemented veneer of pebbles overlying a deep unconsolidated mass of fine clay sediment. The effect of repeated trampling on this sensitive veneer has caused its breakage and collapse into the underlying soft sediments,

65 Chapter 2: Materials and Methods resulting in the formation of deep muddy pools. Parts of the stream substrate remain in original, pristine condition where it crosses underneath sections of passage inaccessible to people.

2.1.3 Sample Collection

Samples were collected from Entrance, Exit and Loons caves, concentrating on three microhabitats; floor sediments, speleothems and moonmilk deposits. Sites were chosen with minimal contamination factors and to reduce impact of our sampling to a minimum. Samples were collected to the side of the main paths to avoid contamination from trampling of cavers and 'clean' speleothems and moonmilk were chosen with no visible human impact or handling

(e.g. mud smears, hand prints etc.). Samples were collected under the provisions of permit number ES 01147 issued by National Parks and Wildlife Service, Tasmania.

Sediment sampling consisted of collecting approximately 10 g/sample using a sterile teaspoon and placing into individual sterile plastic bags. Sterile swabs (EUROTUBO® Collection

Swabs; I.A.S.A) moistened with sterile double distilled water (ddHP) were used to sample from spel~othems. To collect moonmilk deposits, MEl and 3, in Entrance Cave, the upper layer of sediment was scraped away with a sterile teaspoon and sterile 15 mL falcon tubes (REDLINE

Scientific Pty. Ltd.) were inserted into the deposit. The tubes were withdrawn from the deposit approximately half full and capped immediately. Similarly, samples were collected from moonmilk speleothems in Exit Cave, MXl, by inserting sterile 15 mL falcon tubes into the formation till they hit the 'hard' speleothem surface, withdrawing and capping immediately.

Samples of the white mat, ME2, in Entrance Cave were collected by inserting glass slides between the mat and mud or substrate rock. The slides were placed on wet tissue paper within closed petri dishes to keep them hydrated. Samples were transported to the laboratory on ice and stored at 4 °C until processed. Sample locations and descriptions are listed in Table 2.1.

66 Table 2.1: Identity, location and description of samples collected from Entrance, Exit and Loons Caves Sample* Cave and sample location Descnption SEl Entrance Cave, Big Stalagmite Cavern; Dark Zone Dry sediment from indentation, 1.2 ms above floor, Big StalStalagmite.

SE2 '"' Wet sediment from front drainage region of flow form by Big Stalagmite

SLl Loons Cave, "Tarpit", Dark Zone Dry sediment from left hand sidewall deposit above flood zone.

SL2 1111 Wet sediment from bottom of 1 m deep permanent mudhole.

SPE3 Entrance, Big Stalagmite; Dark Zone Swab, droplet on shelf roof, nght hand side of passage.

SPES un Swab, wet flow form, right corner of passage entry.

SPE7 un Swab, Big Stalagmite, dry surface, 1.5 m above floor.

SPElO Entrance, Big Flow form; Twilight Zone Swab, moonmilk mat on cave roof.

SPE12 Entrance, left hand platform; Twilight Zone Swab, old dry flow form.

SPL2 Loons, First Aven; Dark Zone Swab, large dnp stone under aven.

Swab, red droplet on fungal mycelia. SPL3 Loons, dry platform past first Aven; Dark Zone n SPL6 Loons, Lower entrance crawl; Twilight Zone Swab, sloping surface among small stalagmites. g" SPL8 Loons, Sump; Dark Zone Swab, cream flowstone surface. (b" 1-j SPL9 1111 Swab, carrot stalactite. ~

1111 ~ SPL12 Swab, cream flowstone pools. PJ (b" MEl Entrance Cave, Cave Mouth; Light Zone Moonmilk beneath sediment of boulder 1-j...... ,_.PJ ME2 Entrance Cave, Entrance Chamber; Twilight Zone White mat on ceiling mud and rock rJl PJ ME3 Entrance Cave, Second Chamber; Dark Zone Moonm1lk beneath sediment cave floor g, MXl Exit Cave, Ballroom Chamber; Dark Zone Stalactite with thick coating of moonmilk ~ rog 0\ *Samples catalogued using the following code: the first character(s) represent the m1crohabitat (S =sediment, SP = speleothem, M = moonmilk), the last p... '-.:i character represents the cave (E =Entrance, X =Exit, L =Loons). Number 1s indicative of the site that samples were collected from. All samples collected rJl by Jodie van de Kamp and Dr David Nichols, with base support from Dr. Kevin Sanderson during 2001 and 2002 Permit number ES 01147 issued by National Parks and Wildlife Service, Tasmania. Chapter 2: Materials and Methods 2.2 Microscopy and Mineralogy

2.2.1 ESEM and X-Ray Elemental Microanalysis

ESEM was used to visualise microbes within the moonmilk matrix. Fresh, unfixed samples were viewed by ESEM approximately 4 h after collection. Small pieces of moonmilk were removed from the glass slides or falcon tubes using a sterile scalpel blade and placed on aluminium SEM stubs for viewing by ESEM 2020 (Phillips, Australia). The elemental composition of specimens was obtained by means of X-Ray Microanalysis (pers. comm. David

Steele, University of Tasmania, 2002).

2.2.2 X-Ray Diffraction Analysis

Mineralogical compositions of moonmilk were determined by X-Ray Diffraction (XRD)

Analysis. Moonmilk samples were prepared by drying, grinding to <-10-75 µm and pressing into a 25 mm diameter aluminium sample holder. The samples were run on an automated

Philips X-Ray Diffractometer system: PW 1729 generator, PW 1050 goniometer, PW 1710 microprocessor, with nickel-filtered copper radiation at 40 kV /30 mA, a graphite PW 1752 monochromator, sample spinner and a PW 1711 sealed gas filled proportional detector. The PW

1710 system is driven by software packages, "Visual XRD v 2.6" (Diffraction Technology,

Australia) and "PW 1710 for Windows" (CSIRO, Australia), with plotting software, "XPLOT for

Windows" (CSIRO, Australia) and "Traces v 5.1" (Diffraction Technology, Australia).

Interpretation was mostly by manual methods. Samples were calibrated with an internal standard of natural quartz. The semi-quantitative mineralogy was determined by manual search-match methods using a series of prepared standards (pers. comm. Ralph Bottril, Mineral

Resources Tasmania, 2003).

68 Chapter 2: Materials and Methods 2.3 Isolation and Identification of Microbes

2.3.1 Isolation and culturing of microbes

Microbes were isolated from sediments using selective isolation procedures developed by the Antarctic Microbiology Group (University of Tasmania). Approximately 5 g of sediment was transferred into a sterile petri dish and left open in a laminar flow (Gelman Sciences,

Australia) overnight to dry. Sediments were ground to an even consistency using a sterile mortar and pestle and then divided into two equal portions by weight; one untreated control sample (overnight drying and incubation at room temperature for 2 h; OD) and one treated sample (overnight drying and subjected to a heat treatment of 70 °C for 2 h; ODM. Samples were

transferred to individual McCartney bottles containing 9 mL of sterile dd H 20 and placed on a tube roller (Luckham Ltd.) for 30 min to mix.

Microbes were isolated from moonmilk using a modified version of an isolation procedure developed by Olivier Braissant (pers. comm. Universite de Neuchatel, Germany,

2002). Similarly to sediments, up to 5 g of moonmilk (moonmilk being very light in comparison to sediments) was weighed into petri dishes, dried overnight, ground, and divided into four equal portions by weight. Samples were subjected to one of four different treatments by

transferring to individual McCartney bottles containing either: 1) 5% acetic acid (CH3COOH) in

0.01 M MgS04.7HzO; 2) 1% acetic acid in O.OlM MgS04.7H20; 3) 1 mM

Ethylenediaminetetraacetic Acid (EDTA); or 4) 0.1 mM EDTA. Samples were then placed on a tube roller for 30 min to mix.

Dilution series to 10·3 were prepared for sediment and moonmilk samples (initial bottle

10°) and 0.1 mL of each dilution spread plated in duplicate on selective media that favours the growth of actinomycetes; Starch-Casein Agar (SC) (Kuster & Williams, 1964), Arginine-Vitamin

Agar (AV) (Nonomura & Ohara, 1969), Marine Agar (MA) (Oxoid 2216) and R2A Agar (R2A)

(Oxoid CM 906) and non-selective agar for moonmilk samples only; 1/2 strength Tryptone Soya

Agar (1/2 TSA) (Oxoid CM 129) (see Appendix 1 for culture media recipes and preparation).

Swab samples were directly streaked onto the above selective media immediately on return

69 Chapter 2: Materials and Methods from sampling trips. Plates were left to dry in a laminar flow for 30 min. Plates were sealed with

0 2 permeable parafilm (American National Can™, USA) and duplicates .incubated at 25 °C

(within optimal temperature range for isolation of actinomycete) and 10 °C (representing the cave environment) for 2-4 wk or until there was sufficient growth of colonies. After .incubation, actinomycete-like colonies were selected from the primary plates and sub-cultured on Oatmeal

Agar (OA) (Williams & Wellington, 1982) (see Appendix 1). For moonmilk samples, non­ actinomycete-like colonies were also selected and subcultured on 1/2 TSA. Secondary plates were .incubated at 25 °C for approx. 1 wk. All further sub-culturing was conducted as described until pure isolates were obtained. Isolates were cryopreserved (see Appendix 2 for protocol) in replicate for long-term preservation and future use.

2.3.2 165 rRNA gene sequencing and phylogenetic analysis of isolates

2.3.2.1 Extraction of nucleic acids and purification

Genomic DNA was extracted using a method modified from Marmur (1961). Culture biomass was harvested by scraping with a sterile loop. Cells were resuspended in sterile 1.5 mL microcentrifuge tubes (Eppendorf; Greiner Bio-one) with 400 µL saline-EDTA (pH 8) and

1 vortexed (MT 17 Vortex; CHILTERN) to mix. 50 µL of lysozyme (40 mg mL- ; AMRESCO) was added and the tubes .incubated for 30 m.in at 55 °C in a M20 waterbath (LAUDA). 20 µL of

1 proteinase K (10 mg mL· ; SIGMA) was added and the tubes again .incubated for 15 m.in at 55 °C and 20 µL of 25% (w /v) sodium dodecylsulphate (SDS) (SIGMA) for a further 30 m.in at 55 °C.

Tubes were mixed by vortexing between each incubation step. Samples were then subjected to a freeze/thaw step by incubation at-20 °C overnight and thawing at 55 °C for 30 min. Cell debris was separated from aqueous DNA solution by centrifugation at 14000 rpm x 5 m.in, 4 °C in a bench top Eppendorf Centrifuge 5417 R (Laboratory Supply Australia Pty. Ltd.). The supernatant (approx. 400 µL) was transferred to a new sterile microcentrifuge tube. DNA was extracted twice by adding an equal volume of 25:1 (vol/vol) chloroform-isoamyl alcohol

70 Chapter 2: Materials and Methods {SIGMA), followed by vortexing and centrifugation at 14000 rpm x 10 min, 4 °C. The aqueous phase was transferred to a new, sterile microcentrifuge tube each time. DNA was further purified using the Prep-a-Gene® DNA Purification Kit (Bio-Rad) reagents and protocol. DNA products were stored at -20 °C.

2.3.2.2 Agarose gel electrophoresis

To analyse extracted nucleic acids they were fractionated by electrophoresis through 1.0-

1.5% (w /v) agarose (AMRESCO) gels with 0.5 µg/mL ethidium bromide (EtBr) in Tris-acetate

EDTA buffer (40 mM Tris-acetate; 1 mM disodium EDTA; pH 8) {TAE), in a mini-gel apparatus

(Horizon 58, Horizontal Gel Electrophoresis, BRL). 5 µL of DNA product was mixed with 3 µL of 6x gel loading buffer (0.25% bromophenol blue; 0.25% xylene cyanol FF; 40% sucrose) and loaded into the gel. To determine the size of nucleic acid fragments, samples were run alongside

5 µL of the DNA molecular weight marker HyperLadder I (Bioline). Electrophoresis was carried out using a Power Pack 300 power supply (Bio-Rad) at 80 V for 30 min. The DNA/EtBr complex was visualised under short wavelength ultra-violet radiation on an electronic ultraviolet light transilluminator (Ultra. Lum. Inc.).

2.3.2.3 Determination of DNA concentration

The concentration of DNA and PCR solutions (DNAconc) was determined by measuring absorbance at 260 nm using a spectrophotometer (Pharmica) and calculated using the following equation:

1 1 1 1 DNAconc (mg mL- = µg µL- ) = (A260 x 50 µg mL- x D) / 1000 µg mL-

Where D = dilution factor

71 Chapter 2: Materials and Methods

2.3.2.2 165 rRNA gene PCR amplification and purification

The 165 rRNA gene fragment was amplified by Polymerase Chain Reaction (PCR) from

extracted genomic nucleic acids using two universal primers, 10 forward and 1500 reverse

(5tackebrandt et al. 1991) (Table 2.2). These primers were used as they gave thorough coverage

of the three hypervariable regions in the 165 rRNA gene fragment (5tackebrandt et al. 1991). PCR was performed using the Hot5tarTaq™ PCR Master Mix Kit (QlAGEN) reagents and protocol.

PCR reactions consisted of:

Hot5tarTaq Master Mix 25µL Primer 5' (50 pmol) 2µL Primer 3' (50 pmol) 2µL Q-5olution* 2.5 µL Template DNN' __l__g1

ddH20 to total volume 50 µL

* Q-Solution changes the melting behaviour of DNA and was used for PCR reactions that did not work well under standard conditions. A Amount of template DNA added to PCR mix varied depending on the concentration of the DNA, however in most cases 2 µL was sufficient.

PCR reactions were carried out in a PTC - 200 Peltier Thermal Cycler (MJ Research) using the following parameters:

Initial activation step: 15min 95 °C 3-step cycling: Denaturation: lmin 94°C Annealing: lmin 52 °C Extension: 3min 72 °C Number of cycles: 30 Final extension: lOmin 72 °C*

*The final extension step is prolonged to 10 min to allow full extension of any partly amplified DNA fractions.

PCR fragments were purified using the Prep-a-Gene® DNA Purification Kit (Bio-Rad) reagents and protocol. PCR products were electrophoresed as described previously to ensure fragments of the correct size were obtained, and to determine quantity and quality. PCR products were stored at -20 °C.

72 Chapter 2: Materials and Methods 2.3.2.3 165 rRNA gene sequencing

PCR products were sequenced directly using the CEQ 2000 Dye Terminator Cycle

Sequencing (DTSC) Quick Start Kit (Beckman Coulter) reagents and modified protocol. For initial identification of microbes universal primer 519 forward (Stackebrandt et al. 1991) (Table

2.2) was used for amplification. To obtain full sequence information of selected isolates,

universal primers 10 forward and 1500 reverse were also used. Sequence reactions consisted of:

DTCS Quick Start Master Mix 2µL Primer (5 pmol) 1 µL Template PCR* XµL ddHzO to total volume 10 µL

*According to Template Preparation Table in CEQ 2000 DTSC protocol.

Amplification parameters were:

Denaturation: 96 °C 20 sec Annealing: 50 °C 20 sec Extension: 60 °C 4min

Number of cycles: 35

Amplification reactions were purified by ethanol (EtOH) precipitation according to the CEQ

2000 DTCS protocol. Subsequent electrophoresis and analysis was performed using an automated CEQ™ 2000XL Genetic Analysis System (Beckman Coulter). In most cases, 16S rRNA gene fragment sequences spanned nucleotide positions 519-1540 (E.coli equivalent). Entire 16S rRNA gene sequences spanning nucleotide positions 10-1540 were obtained for novel isolates.

73 Chapter 2: Materials and Methods

Table 2.2: Primers used for PCR amplification and sequencmg of 165 rRNA gene fragments.

Pruner Bindmg Primer Sequence (5' to 3') (Reference) Region• 10 (f) 10-29 GAG TIT GAT CCT GGC TCA G (Stackebrandt et al. 1991). 1500 (r) 1520-1540 AGA AAG GAG GTG ATC CAG CC (Stackebrandt et al. 1991). 519 (f) 519-536 CAG CMG CCG CGG TAA TAC (Stackebrandt et al. 1991). 1392 (r) with GC clamp 1406-1392 CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG (Ferris et al. 1996) CCC CAC GGG CGG TGT GTA C 907 (f) 907-926 GGC AGT TAA GGA AAC TCA AA (Santegoeds et al. 1998) pUCIM13 (f) NIA GTA AAA CGA CGG CCA GT (Promega) pUCIM13 (r) NIA CAG GAA ACA GCT ATG AC (Promega)

•Number is based on the Escher1chia coli numbering system from Brosius et al. 1981

2.3.2.4 Phylogenetic Analysis

Sequence electrophoretograms were examined using the program CHROMAS

(http: I /www.technelysium.com.au/chromas.html) in order to resolve any ambiguous base positions. 16S rRNA gene sequences were initially analysed using the National Center for

Biotechnology Information (NCBI) database, Genbank, BLAST tool

(http://www.ncbi.nlm.nih.gov/blast/blast.cgi; Altschul et al. 1997) to identify related sequences available in public databases and to determine phylogenetic groupings of sequences. For phylogenetic analysis, sequences were aligned using the program BioEdit

(http://www.mbio.ncsu.edu/BioEdit/bioedit.html; Hall, 2001) for comparison with validly described and published sequences of representative members of the actinomycete obtained from NCBI GenBank. Distance matrices and phylogenetic dendrograms using the neighbour- joining method were generated using programs DNADIST and NEIGHBOUR of the PHYLIP

3.573c package (Felsenstein, 1993).

74

'' ., Chapter 2: Materials and Methods

2.4 Molecular Analysis of Sediments and Moonmilk

2.4.1 Extraction and purification of nucleic acids from environmental samples

2.4.1.1 Sediments

Total nucleic acids was extracted from sediment following a method modified from

Purdy et al. (1996). Approximately 0.5 g of sediment was aseptically transferred to a 2 mL screw­ cap microcentrifuge tube (Astral Scientific) containing 0.5 g of 0.1 mm diameter zirconia-silica beads (Biospec Products) and suspended in 700 µL 120 mM sodium phosphate buffer (pH 8.0)

(Na2HP04), 500 µL Tris-equilibrated phenol (pH 8.0) (AMRESCO), 50 µL 20% (w /v) SDS and 1

% acid-washed polyvinyl-polypyrrolidone (PVP) (AMRESCO). To lyse the soil microbes, the sample was disrupted in a mini-beadbeater (Biospec Products) at 3 800 rpm for 3 x 30 sec pulses, with a 30 sec incubation on ice between pulses. Cell debris was separated from aqueous DNA solution by centrifugation at 12000 rpm x 2 min, 4 °C. The supernatant was transferred to a new sterile microcentrifuge tube and incubated on ice. The pellet was resuspended in 700 µL 120 mM

Na2HP04 Buffer (pH 8.0) to extract residual nucleic acids from the sample. Cell disruption and centrifugation was repeated as described and the supernatant removed and pooled with the first extraction. Nucleic acids were precipitated by adding 0.1 volumes of 3M sodium acetate (pH 4.6)

(NaOAc) and 2 volumes of cold absolute EtOH followed by incubation at -20 °C for at least 30 min, preferably overnight. The supernatant was removed after centrifugation at 14 OOO rpm x 30 min, 4 °C. The DNA pellet was washed twice in 3~0 µL cold 70% EtOH, with further centrifugation at 14 OOO rpm x 5 min, 4 °C. After removing the supernatant the pellet was

allowed to air dry in a laminar flow hood and subsequently resuspended in 40 µL sterile ddH20.

2.4.1.2 Moonmilk

Nucleic acids were extracted by a procedure developed for this study, modified from

Miller et al. (1999); the Phosphate, SDS, Chloroform-Bead Beater method (PSC-B) (pers. comm.

Susan Turner, University of Auckland, New Zealand, 2003). Approximately 0.5 g of moonmilk was aseptically transferred to a 2 mL screw-cap microcentrifuge tube containing 0.5 g of 0.1 mm

75 Chapter 2: Materials and Methods diameter zirconia-silica beads (Biospec Products) and 300 µL 100 mM Na2HP04 (pH 8.0) and

1 resuspended by vortexing. 30 µL lysozyme (50 mg mL- ) was added and the tubes incubated at

37 °C for 30 min, followed by a further incubation at 65 °C for 60 min to enhance lysis of the cells. Following incubation, 300 µL of SDS lysis buffer (100 mM NaCl, 500 mM Tris pH8, 10%

SDS) was added and the tubes inverted to mix, followed by adding 300 µL chloroform-isoamyl alcohol (24:1; v /v) (SIGMA). Samples were mechanically lysed by bead-beating at 4 OOO rpm for

2 x 40 sec pulses, with a 40 sec incubation on ice between pulses. Cell debris was pelleted from aqueous DNA solution by centrifugation at 12000 rpm x 5 min, 4 °C. The supernatant, approximately 650 µL, was transferred to a new sterile microcentrifuge tube with 360 µL 7M

ammonium acetate (NH40Ac). Tubes were inverted to mix and centrifuged at 12000 rpm x 5 min, 4 °C to separate the phases. The clear supernatant (approximately 580 µL) was transferred to a new sterile microcentrifuge tube and the lower organic phase, with the SDS forming a gel­ like substance, discarded. 0.54 volumes (approx. 315 µL) of isopropanol (SIGMA) was added and the tubes incubated at room temperature for 15 min. After incubation tubes were centrifuged at 12000 rpm x 5 min, 4 °C to pellet the DNA. The supernatant was discarded and the pellet washed twice with 1 mL 70% EtOH, centrifugation at 12000 rpm x 5 min, 4 °C, and the supernatant discarded. The pellet was allowed to air dry in a laminar flow hood before

resuspending in 50 µL sterile ddH20. Additional purification of sediment and moonmilk DNA samples was performed using the CHROMA SPIN™ Columns DNA Purification Kit

(CLONTECH Laboratories Inc.) reagents and protocol. DNA quality and quantity was analysed as described in Section 2.3.2.3.

76 Chapter 2: Materials and Methods

2.4.2 DGGE

DGGE was conducted on four sediment samples and three moonmilk samples (SEl,

SE2, SLl and SL2; ME2, ME3 and MXl; refer to Table 2.1) in accordance with a protocol developed by Powell et al. (2003). A standard control mix consisting of 5 ng µ1·1 each of genomic

DNA extracts from four strains grown routinely in our laboratory and chosen because they denatured at a range of different denaturant concentrations was also used as a control and for comparisons between gels. The 165 rRNA gene fragment was amplified by PCR using the

Advantage® 2 Polymerase Mix (CLONTECH Laboratories Inc.) reagents and protocol with

Universal primers 907 forward (Santegoeds et al. 1998) and 1392 reverse (Ferris et al. 1996) with a

GC clamp (Ferris et al. 1996).

Reactions consisted of:

10 x Buffer 5µL 50 x dNTP Mix (10 mM each) 1 µL Primer 5' (10 pmol) 1 µL Primer 3' (10 pmol) 1 µL 50 x Advantage 2 Polymerase Mix 1 µL Template DNA" ill ddH20 to total volume 50 µL

" Amount of template DNA added to PCR mix varied depending on the concentration of the DNA, however in most cases 1 µL was sufficient.

77 Chapter 2: Materials and Methods The touchdown thermal cycling parameters were:

Initial denaturation step: 5 min 94 °C

1"1 3-step cycling: Denaturation: lmin 94°C Annealing: 1 min 65 °C (decreasing by 1 °C each cycle) Extension: 3 min 72 °C

Number of cycles: 10

znd 3-step cycling: Denaturation: lmin 94°C Annealing: lmin 55 °C Extension: 2min 72 °C

Number of cycles:. 20

Final extension: 4min 72 °C*

*The final extension step is prolonged to 4 min to allow full extension of any partly amplified DNA fractions.

DGGE was conducted using a D-Code Universal Mutation Detection System (Bio-Rad).

Half the volume of PCR products were run on 6% (w /v) acrylamide gels with a denaturing gradient of 20-80% (where 100% dentaurant is 7 M urea and 40% formamide). Gels were run at

80 V for 16 hat 60 °C in 1 x TAE (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA). Standards were run on either side of the gel and the outside lanes were not used. In order to obtain even heat distribution throughout the tank, the entire tank was placed on a magnetic stirring plate.

Gels were stained in 1:1000 Sybergold (Molecular Probes) in the dark with gentle shaking for

approximately 20 min. Gels were washed once with deionised H20 and destained with deionised H 20 for 20 min before viewing on a UV transilluminator (UVP Inc.). Single bands were excised from the gel using a sterile scalpel blade and resuspended in ddH20 in sterile microcentrifuge tube for 16S rRNA gene sequence analysis. Gel photos were scanned in and viewed with the UTHSCSA ImageTool program, developed at the Health Science Centre

(University of Texas, San Antonio, TX, USA) and available on the internet

(ftp:/ /maxrad6.uthscsa.edu). Best banding patterns were obtained by enhancing the contrast and greyscale of the images. The 16S rRNA gene fragment was amplified and purified from

78 Chapter 2: Materials and Methods eluted bands as previously described in Section 2.3.2.4 using the HotStarTaq™ PCR Master Mix

Kit (QIAGEN) reagents and protocol with the exception that DGGE primers 907 (£) and 1392 (r) with a GC clamp were used, and 1 µL of the eluted DGGE band was directly added to the PCR mix. DGGE PCR products were directly sequenced as described in Section 2.3.2.4 using DGGE primer 907 (£) and subjected to phylogenetic analysis as described in Section 2.3.2.5.

2.4.3 Clone Library Analysis

2.4.3.1 165 rRNA gene PCR amplification, ligation and clone library construction.

Clone libraries were generated from four sediment samples and three mo~nmilk samples (SEl, SE2, SLl and SL2; ME2, ME3 and MXl; refer to Table 2.1). The 16S rRNA gene fragment was amplified as described for DGGE analysis (Section 2.4.2) with universal primers,

519 forward and 1500 reverse (Stackebrandt et al. 1991) (Table 2.2). Reactions consisted of:

10 x Buffer 5µL 50 x dNTP Mix (10 mM each) 1 µL Primer 5' (50 pmol) 1 µL Primer 3' (50 pmol) 1 µL 50 x Advantage 2 Polymerase Mix 1 µL Template DNA" ill ddH20 to total volume 50 µL

" Amount of template DNA added to PCR mix varied depending on the concentration of the DNA, however in most cases 1 µL was sufficient.

Thermal cycling parameters were:

Initial denaturation step: 15min 95 °C

3-step cycling: Denaturation: lmin 94°C Annealing: lmin 50 °C Extension: lmin 72 °C

Number of cycles: 30

Final extension: 5min 72 °C*

*The final extension step is prolonged to 5 rnin to allow full extension of any partly amplified DNA fractions.

79 Chapter 2: Materials and Methods PCR fragments were purified using the UltraClean™ PCR Clean-up DNA Purification Kit

(MoBio Laboratories Inc.) and analysed for size and concentration as described in Section 2.3.2.3.

16S rRNA gene PCR frcigments were ligated using the pGEM® -T Easy Vector System I

Kit (Promega) reagents and protocol. Ligation reactions were subjected to an overnight incubation at 4 °C to produce the maximum number of transformants. Transformation of ligation products was performed using the Epicurian Coli® XL2-Blue Ultracompetent Cells

(Stratagene) reagents and protocol. Transformants were screened using blue-white colony colour selection. Aliquots (50 µL and 100 µL) of the transformation mixture were plated on Luria

Broth agar plates containing 100 µg mL·1 ampicillin (SIGMA) (LB-Amp) and coated with 100 µL

1 0.1 M iso-propyl-beta-D-thio-galactopyranoside (120 mg mL- ) (IPTG) (SIGMA) and 20 µL 5-

1 bromo-4-chloro-3-indoyl-beta-D-thio-galactopyranoside (50 mg mL- ) (X-gal) (SIGMA) (see

Appendix 1). Plates were incubated overnight for 16-20 hat 37 °C. Colonies containing recombinant plasmids with the 16S rRNA gene fragment appear white, whereas colonies containing un-recombinant colonies appear blue. Appr~ximately 150 white colonies from each library were sub-cultured to LB-Amp plates and re-incubated overnight at 37 °C.

2.4.3.2 Restriction Fragment Length Polymorphism screening and 165 rRNA gene sequencing of clones

Recombinant plasmids were extracted and purified from transformed cells using the

UltraClean™ Mini Plasmid Prep Kit (Mo Bio Laboratories Inc.) reagents and protocol. Plasmids were electrophoresed in a 1 % (w /v) agarose gel, 80 V x 40 min (see Section 2.3.2.2) to confirm they contained the 16S rRNA gene insert. Recombinant plasmid DNA were confirmed by correlation of their position on the gel with a plasmid known to contain the correct size insert.

Plasmids containing an insert of the correct size were further screened by Restriction Fragment

Length Polymorphism (RFLP) analysis. Restriction digests were performed on plasmids by separate incubation with the restriction nucleases Hinfl (New England Biolabs) and Rsal (New

England Biolabs) and accompanying buffers (New England Biolabs) at 37 °C for 3-4 h. Digests were fractionated by electrophoresis on 3% (w /v) agarose gels, 100 V x 3 h, (see Section 2.3.2.2) resulting in characteristic banding patterns allowing the diversity and abundance of cloned

80 Chapter 2: Materials and Methods phylotypes to be approximated. Clones exhibiting diverse banding patterns (including 2-4 duplicate clones possessing the same RFLP pattern) were selected at random for sequencing.

Clones were sequenced as described in Section 2.3.2.4 with the exception that plasmid templates were subjected to a pre-heat treatment and primers pUC/Ml3 forward and reverse were used for amplification (Promega) (see Table 2.2). Binding sites for these primers are located on the pGEM® -T Vector, positioned either side of the insert. The pre-heat treatment consisted of diluting the template with water to the appropriate concentration, heating to 96 °C for 1 min in a

PTC - 200 Peltier Thermal Cycler (MJ Research), and cooling to room temperature before adding the remainder of the sequencing-reaction components. In most cases, 16S rRNA gene clones were entirely sequenced with the sequences spanning nucleotide positions 519 - 1540 (E. coli equivalent).

2.4.4 Phylogenetic and biodiversity analysis

Phylogenetic analysis was conducted as described in Section 2.3.2.4 with the exception that the Ribosomal Database Project II (RDP) CHIMERA-CHECK program

(http:/ /rdp.cme msu edu/; Maidak et al. 2001) was used to detect PCR-amplified hybrid sequences. In addition, potential chimeras were determined from inconsistencies in branching order. Chimerical clones detected were not included in subsequent phylogenetic or biodiversity ' analyses. For calculation of diversity indices, the libraries were normalised to 50 clones using the rarefaction method (Simberloff, 1972) by utilising the program RAREFACT.FOR written by C. J.

Krebs (University of British Columbia) and which is available through the internet

(http://www2.biology.ualberta.ca/jbrzusto/rarefact.php).

81 Chapter 2: Materials and Methods Estimates of Diversity (H) were determined using the Shannon-Weaver (or Shannon-

Weiner) Index (Krebs, 1989). H' is given by the formula:

k H' = n log n - L N log N i=l n

where k is the total number of unique phylotypes, n is the total number of clones and N is the

number of observations of each phylotype (i).

Measures of dominance concentration were determined using the Simpson Index (SI)

(Krebs, 1989). SI' is given by the formula:

k SI'= L Ni ( Ni - 1 ) i = 1 n(n-1)

Equitability indices (JJ were based on Shannon-Weaver index data. J' is given by the

formula:

]'= H' Hmax

Where H max is equal to log k.

Biodiversity coverage (C) (Mullins et al. 1995) was derived by the formula:

C=l-l!!J N

Where ni is the number of phylotypes containing only one clone, and N is the total number of clones.

Pairwise comparisons of clone libraries were carried out using the Similarity Coefficient

(S) (Odum, 1971). Sis derived from the formula:

S=2C A+B

Where A and B are the number of phylotypes in libraries A and B respectively, and C is the number of shared phylotypes.

82 Chapter 3: Results and Discussion

Chapter 3: Results and Discussion

3.1 Microscopy and Mineralogy

Putative moonmilk samples were collected from an extensive speleothemic deposit in the dark zone of Exit Cave (MXl) and white mat-like material on the ceiling rock of Entrance

Cave within the twilight zone (ME2). During the course of this study large moonmilk-like deposits were found beneath sediment in Entrance Cave (MEI, ME3) and analysed for comparison. ESEM with X-Ray Microanalysis was employed to investigate the microbe-mineral interface of moonmilk samples.

The samples collected from the ceiling of Entrance Cave (ME2) exhibited distinct, isolated areas of thin white material on the muddy rock surface (Figure 3.2; A). X-Ray microanalysis of this material (Figure 3.1; A, B) revealed high levels of silicon and aluminium suggesting a day-type mud and high levels of carbon and oxygen suggesting areas of organic material. Figure 3.2 (B) illustrates that the isolated areas of white material on the mud surface from ME2 contained a crystalline character associated with biological growth of hyphal material.

In the dark zone of Lechuguilla Cave CaC03-mineralised organic filaments have been reported

(Cunningham et al. 1995). High magnification of the mat demonstrated the presence of hyphae­ forming microorganisms with segmented hyphae of width 0.5-1 µm, consistent in size and morphology with filamentous actinomycetes. Non-biological (crystalline) structures were evident both beneath the mat of hyphal growth and also encrusting individual hyphae. Putative cells and an organic matrix can be frequently seen in moonmilk samples with SEM or in thin sections, but not in all cases (Northup et al. 2000). Biological material or cells were not evident in

ESEM analysis of moonmilk samples ME3 or MXl, though CYBR staining confirmed the presence of DNA in the samples (data not shown).

83 Chapter 3: Results and Discussion

Si

0 0

a

Figure 3.1: X-Ray microanalysis spectra of sample ME2 from the ceiling rock in the twilight zone of Entrance Cave. (A) Spectra of mud containing high levels of silicon, oxygen and aluminium consistent with a clay-type mud. (B) Spectra of mat sample ME2, illustrating the presence of organic material, indicated by high levels of carbon and oxygen.

For the purpose of distinguishing mondmilch from other carbonate speleothems,

Fischer (1988) defined true calcite moonmilk as a calcite microcrystalline or needle-crystalline speleothem with a minimum calcite content of 90 % weight. ESEM of samples MXl and the crystalline areas of ME2 (Figure 3.3) shows the needle-fibre crystalline characteristics of the calcite (confirmed by X-Ray microanalysis, data not shown). XRD studies revealed that the mineralogical composition of moonmilk samples from both Entrance Cave and Exit Cave were almost identical (Table 3.1). Moonmilk samples consisted predominantly (85-100%) of calcite

(Ca03), with trace amounts of quartz, mica (clay, most likely illite) and hydrated iron oxide, goethite [oc-FeO(OH)].

Table 3.1: X-Ray Diffraction analysis of moonmilk samples from Entrance Cave, ME2 and ME3, and Exit Cave, MXl. Approximate mineralogy recorded as % weight.

Sample Calcite Quartz Mica* Goethite

ME2 85 3 10 2

ME3 100

MXl 100

•Probably illite. Note: Peak overlap may interfere with identifications and quantifications. Minerals present in trace amounts may not be detected.

84 Chapter 3: Results and Discussion

Figure 3.2: Photographs and ESEM pictures of cave sample ME2 showing biological hyphal material and calcite encrusted hyphae. (A) White mat with reflective droplets on ceiling of Entrance Cave (ME2). B-F: ESEM images of sample ME2. (B) Mat of microbial growth on the ceiling. (Q Oumps of hyphae encrusted with calcite and uncalcified hyphae on the surrounding mud. ESEM images of calcite encrusted microbial filaments at high magnification. (D) Detailed view illustrating different degrees of encrustation exhibited by hyphae. (E) Detailed view of calcite encrusted hyphae. (F) Segmented hyphae, width approx. 0.5-1 µrn.

85 Chapter 3: Results and Discussion

Figure 3.3: ESEM images of moonmilk samples illustrating microcrystalline, needle-fibre form of CaC03 crystals (confirmed by X-Ray microanalysis, data not shown). (A) Sample ME2 from Entrance Cave. (B) Sample MXl from Exit Cave.

XRD and ESEM results indicate that samples MEI, 3 and MXl are true calcite moonrnilk

(98-100% CaC03). Sample ME2 had a slightly lower calcite composition (85%). The thin, mat-like nature of this sample from the ceiling of Entrance Cave made it difficult to collect samples from just the white material and inevitably some of the clay layer (2-3mm thick) on the ceiling was collected too, perhaps accounting for the higher clay content (10 %) of this sample

3.2 Method Development for Calcite Moonmilk Samples

It has previously been suggested that DNA extraction from environmental samples

containing high levels of CaC03 is problematic (Guthrie et al. 2000; Northup, pers. comm. 2001).

Initial clone analysis of samples ME2, ME3 and MXl, that have high calcite content (85-100%) as demonstrated by XRD analysis, (Section 3.1) resulted in a single phylotype most closely related to y-Proteobacteria, Pseudomonas fluorescens. Isolations also proved to be problematic initially producing almost pure cultures of Bosea thiooxidans. Though these organisms were dominant components of the calcite-based microbial communities (Table 3.2), ESEM results depicting hyphal organisms indicated that these results were not necessarily representative of the true diversity. DNA extraction methods and cultivation procedures rely on the bacterial cells being readily released from their environmental matrix. Current DNA extraction protocols for

86 Chapter 3: Results and Discussion molecular analyses are poorly adapted for lithic or encrusted microbial communities due mostly to the hard, usually cemented nature of the mineral matrix (Wade & Garcia-Pichel, 2003).

Guthrie et al. (2000) suggested that as DNA was released from coral matrices it was adsorbed by the calcite minerals resulting in very low quantities of DNA being recovered. A significant portion of this study was directed at method development to enhance DNA extraction and cultivation procedures for calcite cave samples.

A comparison of three DNA extraction protocols was undertaken: a modified protocol from Purdy et al. (1996) utilised for cave sediments in this study, the protocol from Guthrie et al.

(2000) which was successful for coral samples, and a modified protocol from Miller et al. (1999), the Phosphate, SDS, Chloroform-Bead Beater method (PSC-B) which was successful with pure opal-A silica sinter samples (pers. comm. Dr. Susan Turner, University of Auckland, 2003).

DGGE analysis of PCR amplified 165 rRNA gene from DNA product of the three extraction protocols was used to determine which method was most appropriate. PCR product resulting from the modified PSC-B DNA extraction displayed the highest degree of diversity in the banding pattern for all samples (Figure 3.4). Thus this protocol was utilised for further clone library analysis.

35%

65%

ME3 ME2 ME3 ME2

Figure 3.4: 165 rRNA gene DGGE community fingerprint of Entrance Cave moonmilk samples, ME2 and ME3. (A) DNA extraction using PSC-B method. (B) DNA extraction using Guthrie et al. (2000) coral method. Illustrates the greater diversity of banding patterns for DNA extracted using PSC-B method.

87 Chapter 3: Results and Discussion Microbes were isolated from moonmilk using a modified version of an isolation procedure developed by Olivier Braissant (pers. comm. Universite de Neuchatel, Germany,

2002). Calcite samples were subjected to one of five different treatments to dissolve the carbonate and free bacterial cells for cultivation:

1) 5% acetic acid (CH3COOH) in 0.01 M MgS04.7H20

2) 1% acetic acid in O.OlM MgS04.7Hz0

3) 1 mM Ethylenediaminetetraacetic Acid (EDTA)

4) 0.1 mM EDTA

5) ddH20 (control)

The 1% acetic acid and lmM EDTA treatments produced the greatest number of different colony

morphology types on primary isolation plates (data not shown). The 0.1 mM EDTA and ddH20 treatments resulted in far fewer colonies and only a limited number of colony morphologies on isolation plates, indicating that these treatments did not sufficiently separate the cells from the calcite matrix. The 5% acetic acid treatment produced the least number of colonies on primary plates possibly due to the acid being bacteriocidal at this concentration. It is recognised that the application of EDTA and acetic acid solutio.ns may have introduced unknown degrees of bias to the resulting isolations. However, as it was necessary to dissolve the carbonate to obtain greater diversity in isolations, this bias was unavoidable.

88 Chapter 3: Results and Discussion

3.3 Phylogenetic Diversity Overview

165 rRNA gene clone libraries were constructed from D~\JA extracted from four sediment samples (SEl, SE2, SLl and SL2) and three moonmilk samples (ME2, ME3 and MXl).

Libraries were constructed with universal primers. Approximately 100-120 clones per library were screened by analysis of RFLP patterns and selected representatives of novel RFLP patterns were sequenced. Sequences greater than 500 base pairs (bp) were included in phylogenetic analysis. Groups of two or more highly related sequences (~ 98% sequence identity) were considered to belong to the same sequence type designated a phylotype. From a total of 488 nonchimeric clones analysed from seven libraries, 148 phylotypes were defined affiliated with the domain Bacteria. A total of 43 phylotypes were found in two or more libraries. Table 3.2 provides a summary of the representative sequences and their phylogenetic affiliations. The majority of clones fell into three major phylogenetic groups: the Proteobacteria (dominating all samples), the high G+C Gram-positive Actinobacteria, and the Cytophaga-Flavobacterium­

Bacteroides (CFB) group. DGGE and subsequent 165 rRNA gene sequencing of bands was used to analyse moonmilk samples for comparison to clone library results. DGGE was also applied to sediment samples however the greater species diversity from sediments made the accurate defining of individual bands for sequencing difficult, a common result for sediment samples.

Common banding patterns between samples indicate common community representatives. A total of six major bands present in moonmilk samples were sequenced and phylogenetically aligned with the a-Proteobacteria, Actinobacteria and CFBs.

Cultures were isolated from four sediment and three moonmilk samples and from swabs of speleothems in Entrance and Loons Caves to investigate culturable diversity (see Table

2.1 for sample locations). Sediment sites were chosen away from main pathways in the dark zone of the caves and covering a range of sediment types from two caves of different character:

Entrance Cave (SEl - dry sediment, SE2 - saturated sediment) and Loons Cave (SLl - dry sediment, SL2 - saturated sediment). Moonmilk samples were chosen to include two cave types

(Entrance and Exit) and cover a range of forms, speleothemic (MXl), mat-like (ME2) an~ floor

89 Chapter 3: Results and Discussion deposit (ME3). Selective procedures and media favouring actinomycete growth were applied to sediment and speleothem samples whereas non-selective procedures and media were used for moonmilk samples. Gross morphology was used to discard duplicate cultures and isolates displaying novel morphology were identified using 165 rRNA gene sequencing and phylogenetic analysis. Groups of two or more highly related sequences (:::: 97.5% identical) were considered to belong to the same species in accordance with the definition of a bacterial species

(Goebel & Stackebrandt, 1994; Vandamme et al. 1996). A total of 302 isolates belonging to 39 genera were sequenced, mostly belonging to the order Actinomycetales. Table 3.3 summarises the phylogenetic affiliations of representative isolates. The majority of actinomycete isolates from all samples belonged to the Streptomycineae, Pseudonocardineae, Corynebacterineae and Micrococcineae.

Isolates from moonmilk samples belonged to the Actinomycetales, Firmicutes, Proteobacteria and

CFB groups.

90 Cha2ter 3: Results and Discussion Table 3.2: Summary of Phylotype* Abundance and Phylogenetic Affiliations from Cave Microhabitats PHYLOTYPEA NEAREST TAXON (% IDENTITY)8 ABUNDANCE IN MICROHABITATc SEl SE2 SLl SL2 ME2 ME3 MXl D BACTERIA a-Proteobacteria Caulobacterales Caulobacteraceae ME30021 Nztrobacteria hamadamenszs AY569007 (93.9%) 1 MON0045 Brevundzmonas alba AJ227785 (94.6%) 1 1 CAVOOOl Brevundzmonas alba AJ227785 (98.2%) 7 1 2 DMONl Brevundzmonas alba AJ227785 (98.2%) -./ -./ -./ Rhizobiales Beijerinckiaceae MX10051 Methylocella palustrzs Y17144 (89.1%) 4 Bradyrhizobiaceae ME20020 Bradyrhzzobzum japomcum AF363150 (98.2%) 3 CAV0002 Bosea thiooxzdans X81044 (99.8%) 2 4 14 1 DMON2 Bosea thzooxzdans X81044 (99.8%) -./ -./ MX10048 Bosea thzooxzdans X81044 (90%) 1 5120043 Afipm masszlzenszs AY029562 (95.1 %) 2 MX10021 Afipm genosp 9 U87780 (99.2%) 1 CAV0008 Rhodopseudomonas palustrzs D12700 (93.7%) 2 2 1 Brucellaceae 5120011 Ochrobactrum anthropz U70978 (94.2%) 1 Hyphomicrobiaceae ME20041 Hyphomzcrobzum sulfomvorans AF235089 (93.8%) 1 5ED0019 Hyphomzcrobium vulgare X53182 (91.9%) 1 1 1 5110054 Devosza rzboflavma AY512822 (99.6%) 1 2 Methylobacteriaceae 5E10044 Methylobacterzum extorquens 120847 (91.1 % ) 1 Phyllobactenaceae 5E20001 Phyllobacterium myrsznacearum AJ011330 (99.2%) 2 1 1 ME20015 Ammobacter mzgataenszs AJ011761 (96.1 %) 1 Rhizobiaceae MX10017 Rhizobium gzardmn U86344 (99.1 %) 1 Rhodobacterales Rhodobacteriaceae 5120056 Rhodobacter azotoformans D70846 (97%) 1 MX10016 Rhodobacter sphaeroides D16424 (98.4%) 1 5E10043 Amarzcoccus macauenszs U88042 (85.7%) 1 5PE008 Paracoccus solventzvorans AY014175 (??%) Sphingomonadales 5E10056 Sphingomonas aerolata AJ429240 (97.5%) 1 MON0003 Sphmgomonas phyllosphaerae AY453855 (97.1 %) 1 1 CAV0009 Sphingopyxzs alaskensisAF378795 (94.2%) 1 2 2 5 1 DMON3 Sphzngopyxzs alaskenszsAF378795 (93%) -./ -./ f3-Proteobacteria Burkholderiales Acaligenaceae 5120039 Derxza gummosa (91.6%) 1 3 5E20024 Bordetella pertussis AF366576 (91.4%) 1 Burkholderiaceae 5110008 Burkholderia sordidicola AF512827 (92.9%) 4 5110014 Limnobacter thiooxidans AJ289885 (89.2%) 1 2 CAV0003 Pandorea apzsta AF139172 (93.1 %) 2 1 1 Commamonadaceae CAV0004 Hydrogenophaga defluvzi AJ585993 (94.3%) 1 2 1 5120003 Hydrogenophaga palleromz AF019073 (98.7%) 1 MX10008 Delftia tsuruhatenszs A Y302438 (97.3%) 1 MONOOlO Polaromonas vacuolata U14585 (95 7%) 1 3 5110033 Varzovorax paradoxus AJ420329 (99.3%) 1 5120010 Aczdovorax valerzanellae AJ431731 (96.1 %) 1 5E20028 Ottowia thzooxydans AJ537466 (92.4%) 1 Oxalobacteraceae CAV0005 Janthmobacter agarzczdamnosum Y08845 (98.3%) 2 2 4 3 MON0015 Masszlza timonae U54470 (97.5%) 5 4 CAV0006 Duganella vzolaceusniger AY376163 (97%) 1 5 5 ME30010 Oxalobacter formzgenes U49758 (96 3%) 1 1 CAV0021 Herbaspirzllum f!:zsmgense AJ238358 (??) 2 3 1

Continued on next page 91 Cha12ter 3: Results and Discussion

PHYLOTYPEA NEARESTTAXON (% IDENTITY)8 ABUNDANCE IN MICROHABITATc SEl SE2 SLl SL2 ME2 ME3 MXl D Hydrogenophilales Hydrogenophilaceae 5110010 Thzobaczllus denitrificans AJ243144 (94.5%) 2 Methylophilales Methylophilaceae SL00008 Methylophilus leismgerz AF250333 (97.8%) 2 5 SE20011 Methylophilus freyburgenszs AJ517772 (93.3%) 8 SL00038 Methylovorus mays AY486132 (94.3%) 3 8 Nitrosomonadales Nitrosomonadaceae SL20020 Nitrosospira brzensis AY123800 (90.2%) 4 Unclassified SED0039 Thzobacter subterraneus AB180657 (89%) 1 1

&-Proteabacteria Desulfuromonadales Desulfuromonadaceae MON0018 Desulfuromonas thiophzla Y11560 (90%) 1 1 1 Geobacteraceae SE10098 Geobacter pelophzlus U96918 (91 % ) 1 Desulfoarculales Desulfoarculaceae SL0043 Nztrospma gracilis L35504 (92%) 2 2 1

y-Proteobacteria Acidithiobacillales Acidothiobacillaceae SE10004 Aczdothiobacillus ferroxzdans AJ457808 (98%) 2 Alteromonadales Alteromonadaceae SED0012 Marmo bacterium georgzense AB021408 (99%) 1 1 Chromatiales Chromatiaceae SED0017 Nztrosococcus oceanz AF363287 (91 % ) 3 1 SL10022 Nztrosococcus oceani AF363287 (90%) 1 SEDOOlO Thzocapsa roseoperscma Y12303 (93%) 3 1 SE10089 Thzobaczllus prosperus AY034139 (94%) 1 Ectothiorhodospiraceae SE10058 Thioalkalivibrio thzocyanodenztrzficans AY360060 6 (92%) Enterobacterales SED0008 Photorhabdus luminescens D78005 (95%) 9 9 2 Legionellales SE10003 Legzonella londmzenszs Z49728 (94%) 1 Methylococcales SE10021 Methylococcus capsulatus X72770 (90%) 1 ME30011 Methylococcus capsulatus X72770 (91 %) 1 Pseudomonadales CAVOOll Pseudomonas fluorescens AF094729 (98%) 1 3 2 4 3 SE20012 Pseudomonas putida AF094743 (98%) 2 SE20021 Pseudomonas angu1!11sept1ca X99540 (98%) 1 MX10050 Uncultured bacterium clone Cll-Kll AJ421116 1 (95%) Moraxellaceae ME30060 Acinetobacter 7ohnsoni1 Z93440 (94%) 2 Thwtrichales Thiotrichaceae SE20006 Achromabum oxahferum L48227 (93%) 1 Xanthomonadales SE10045 Lysobacter gummosus AB161361 (97%) 2 SE10044 Lutezmonas mephitzs AJ012228 (97%) 1 SL10051 Frauterza aurantza AJ010481 (95%) 1 SED0009 Hydrogenocarbophaga effusa AY363244 (93%) 1 2 1 CAV0030 Pseudoxanthomonas mexzcana AF273082 (96%) 1 1 1

Actinobacteria Actinobacterideae Actinomycetales Corynebacterineae ME20019 Nocardza carnea X80607 (99%) 3 2 2 ME20104 Nocardza corynebacterozdes X80615 (94%) 2 Micrococcineae MEX005 Arthrobacter chlorophenolzcus AF102267 (96%) DMON4 Arthrobacter chlorophenolzcus AF102267 (96%) -./ -./ -./ CAV0027 Arthrobacter pascens X80740 (99%) 1 2 2 2 CAV0046 Arthrobacter ox't.dans X83408 (99%) 1 2 Conbnued on next page. 92 Cha£ter 3: Results and Discussion

PHYLOTYPEA NEAREST TAXON (% IDENTITY)8 ABUNDANCE IN MICROHABITATc SEl SE2 SLl SL2 ME2 ME3 MXl D SL20016 Arthrobacter psychrolactzcus AF134183 (98%) 1 MON021 Knoellza subterranea AJ294413 (99%) 3 1 1 2 Pseudonocardineae ME20021 Pseudonocardza asaccharolytzca Y08536 (95%) 3 SL10013 Actznobispora alamniphila AF325726 (96%) 1 ME20012 Amycolatopszs fastzdiosa AJ400710 (96%) 1 ME20103 Amycolatopszs sulphurea AJ293756 (96.5%) 2 ME20081 Saccharothrix coeruleofusca AF114805 (95%) 1 CAVOOlO Saccharothrix texasenszs AF350247 (97%) 2 1 1 4 MON0007 Saccharothrix cryophilzs AF114806 (95%) 4 2 3 DMON5 Saccharothrix cryophilis AF114806 (95%) -.J -.J -.J MON0018 Lentzea albidocapillata X84321 (96%) 1 SE10086 Lechevalierz aerocolonzgenes AY196703 (88%) 1 SL20046 Kzbdelosporangium phillipznense AJ512464 (95%) 1 Propionibacterineae CAV0023 Propionzbacterium acnes AB042288 (98%) 1 1 SED0051 Nocardioides fulvus AF005016 (94%) 1 2 MX10002 Nocardioides sp. LMG20237 AJ316318 (92%) 1 MX10032 Pzmelobacter simplex 278212 (98%) 1 Micromonosporineae SL10009 Micromonospora echznoaurantzaca X92618 (98%) 1 ME20061 Actznoplanes cyaneus AB036997 (95%) 1 MX10039 Virgosporangzum ochraceum AB006167 (91 % ) 2 Frankineae SE0098 Frankza sp. 2 2 SE10039 Blastococcus saxobsuiens AJ316570 (90%) 1 Streptomycineae SL10019 Streptomyces caviscabies AF112160 (99%) 2 1 1 ME20022 Streptomyces subrutilis X80825 (97%) 1 2 ME30039 Streptomyces sangl1er1 AY094364 (98%) 1 ME20033 Streptomyces vwlaceoruber AF503492 (98%) 1 SE00050 Streptomyces yunnanenszs AF346818 (91 %) 1 1 CAV0015 Streptomyces macrosporus 268099 (90%) 1 2 ME20041 Streptomyces rutgersenszs 276688 (99%) 1 SE10028 Streptomyces galzlaeus AB045878 (98%) 1 SL10055 Kitasatospora medwcidzca U93324 (97%) 1 Rubrobacterideae ME30059 Thermoleophilum minutum AJ458464 (89%) 1 ME30009 Thermoleophzlum album AJ458463 (93%) 1 Sphaerobacterideae SE10001 Sphaerobacter thermophilus AJ420142 (90%) 1 Unclassified Actinobacteria SE10060 Cand1datus Mzcrothrzx parvzcella X89774 (91 %) 2 1

Firmicutes SL10009 Ruminococcus flavefaczens X85097 (91 %) 2 MX10063 Bacillus subtilus AB042061 (99%) 1 1 ME20098 Sporosarczna ureae AF202057 (98%) 2

Cytophaga-Flavobacteria-Bacteroides Bacteroidetes Flavobacteriales Flavobacteriacea MX10045 Cryomorpha ignava AF170738 (92%) 1 SL10003 Flavobacterza ferrugzneum M62798 (96%) 1 SL10020 Flavobacterium columnare M58781 (93.3%) 1 CAV0015 Flavobacterza lzmicola AB075230 (98%) 1 2 6 8 ME30007 Flavobacterza lzmzcola AB075230 (93%) 2 CAV0018 Flavobacterza leeana AB180738 (98%) 2 3 1 1 10 DMON6 Flavobactena leeana AB180738 (98%) -.J -.J CAV0030 Antarctic bacterium R-7933 AJ440987 (97.1 %) 1 1 Sphingobacteriales Sphingobacteriaceae MON0015 Pedobacter cryconztzs AJ438170 (97%) 2 1 ME30041 Sphzngobacterzum faeczum AJ438176 (93.4%) 3 Bacteroidales

Continued on next page. 93 Cha12ter 3: Results and Discussion

PHYLOTYPEA NEARESTTAXON (% IDENTITY)8 ABUNDANCE IN MICROHABITATc

SEl SE2 SLl SL2 ME2 ME3 MXl D SL0019 Flexibacter tructuosus M5S7S9 (92%) 1 1 SL10002 Uncultured bacterium clone C44K17 AJ297617 1 (92.9%) CAV0026 Bacteroidetes bacterium Mo-0.2plat-K3 AJ622SSS 1 1 1 (90.9%)

Acidobacteria ME20061 Uncultured bacterium DADOS Y12597 (97%) 1 MON0045 Uncultured bacterium DADOS Y12597 (94%) 2 2 ME20013 Uncultured bacterium DADOS Y12597 (90%) 2 ME20020 Bactenum Ellin6075 AY234727 (94%) 1 ME20050 Bacterium Ellm6075 A Y234727 (91 %) 1 SL20005 Bacterium Ellin6071 AY234723 (95%) 1 SL10020 Bactenum Ellin52S9 AY234640 (S9%) 1

Planctomycetales SE10039 Planctomyces braszlzenszs AJ231190 (90%) 5 CAV0062 Planctomyces marzs AJ231184 (90.4%) 4 2 1 1 SED0061 Pzrellula staleyz AJ2311S3 (96%) 2 1 2 2 SE0051 Pzrellula sp. XS1947 (SS S%) 3 1 SED0047 Planctomycete str.292 AJ2311S2 (S7.2%) 2 1 1 SL10061 BacteriumDR2A-7G19 AB127S5S (91.2%) 1 ME30013 Gemmata-like str. C1uq14 AF239693 (Sl.9%) 2

Chloroflexi (green nonsulfur) ME20011 Caldilznea aerophzla AB067647(S6.4%) 2 SE0037 Caldilznea aerophzla AB067647 (90%) 1 1 MX10044 Dehalococcoides ethenogenes AF00492S (95.6%) 1 MX10041 Anaerolznea thermophzla AB046413 (91.9%) 1 ME30006 Urudentified bacterium strain BD3-16 AB015556 1 (S6.9%)

Verrucomicrobia SE10094 Uncultured verrucomicrob1um DEVOlO 2 AJ401127 (92%); Verrucomzcrobza spinosum X90515 (S6.5%) SE10006 Opitutus sp VeSm13 X99392 (91.S%) 1

OP10 SL20004 Uncultured bactenum SJA-176 AJ009504 (S6%) 1 SL20017 Uncultured bacterium GC55 AJ27104S (90.2%) 1 Gemrnatimonadetes SL20096 Gemmatzmonas aurantiaca AB072735 (S7 6%) 3 SL20036 Bactenum Ellm 5301 AY234652 (S7.S%) 2

ARCHAEA Crenarchaeota SL20017 Uncultured archaeon WSB-11 AB055993 (91.S%) 1 Desulf!!:.rococcus amy_loly_tzcus AF250331 (75%) Total:# phylotypes 40 34 39 39 31 29 40 (#clones) (72) (60) (68) (68) (71) (61) (75)

*Phylotypes represented m sed!IIlent and moommlk samples (CAV) Phylotypes represented in sediments of Entrance and Loons Caves (SED). Phylotypes represented m more than one moonmilk sample (MON) DGGE bands from moonmilk (DMON); presence(.../), absence(-). A A umque sequence or group of highly related sequences (> 98% identical) cous1dered to belong to the same sequence type. 8 Inferred from drrect sequence comparison to representative sequences on GENBANK. Access10n numbers given c No of clones m phylotype represented m each nucrohabitat studied based on drrect sequence comparisons or inferred from RFLP patterns 0 Microhabitats represented by samples. SE! (dry sediment, Entrance Cave), SE2 (wet sediment, Entrance Cave), SL! (dry sediment, Loons Cave), SL2 (wet sediment, Loons Cave), ME2 (calcite mat, Entrance Cave), ME3 (moonnulk, Entrance Cave), and MXl (moonmlik, Exit Cave).

94 Chapter 3: Results and Discussion Proteobacteria

The Proteobacteria were the most commonly sampled group (35.2-76.5% of clones) present within the cave samples. Representatives of the alpha (a), beta(~), gamma (y) and delta

(8) subclasses were detected in varying proportions in the clone libraries. No epsilon (c)

Proteobacteria clones were detected in this study.

a-Proteobacteria

A total of 81 clones representing 24 phylotypes were affiliated with the a-Proteobacteria and representatives were detected in all libraries (Table 3.2). Three DGGE bands (DMONl, 2 and 3) affiliated with the a-Proteobacteria were present in all 3 moonmilk samples. Isolates from sediment, speleothems and moonmilk were also affiliated with the a-Proteobacteria. Figure 3.5 displays an evolutionary distance dendrogram of representatives of the a subclass and associated cave clones, DGGE bands and isolates.

The most pronounced clade was the Rhizobiales consisting of 15 phylotypes from all samples affiliated with Beijerinckiaceae, Bradyrhizobiaceae, Brucellaceae, Hyphomicrobiaceae,

Methylobacteriaceae, Phyllobacteriaceae and the Rhizobiaceae. The most dominant phylotype present in high numbers in both Loons sediments and all moonmilk samples (CAV0002) was most closely related to Bosea thiooxidans (99.8% sequence similarity), a thiosulfate oxidiser (Das et al.

1996). The isolation of a strain of Bosea thiooxidans and the presence of 14 clones of this phylotype in the Entrance mat material indicates that this is a major component (19.71 %) of the total microbial community. The Bosea thiooxidans phylotype was also detected in all moonmilk samples by DGGE analysis (band DMONl). A number of clones were affiliated with methylotrophic taxa (phylotypes MX10051, ME20041, SED0019, SE10044) including representatives of the genera Methylobacterium, Methylocella, and Hyphomicrobium. Phylotypes

ME20041 and SED0019 formed a deep lineage within the Hyphomicrobiaceae. A novel pink­ pigmented Methylobacterium sp. was isolated from moonmilk and phylotypes branching with genera Methylobacterium and Methylocella were detected in Entrance sediment and moonmilk from Exit. Phylotype MX10051, consisting of four clones, formed a deep branching lineage

95 Chapter 3: Results and Discussion within the Beijerinckiaceae affiliated loosely with Methylocella paulstris (89.1 % sequence similarity). M. palustris is a methanotrophic acidophile isolated from peat wetlands (Dedysh et al. 2003). Other members of the Bezjerinckiaceae are free-living aerobic nitrogen-fixing bacteria

(eg. Beijerinckia) which grow well in acidic soils. Sediment phylotypes were also affiliated with nitrogen-fixing bacteria including those usually associated with plant nodules (eg. Rhizobium,

Bradyrhizobium) (Young & Haukka, 1996).

The second clade of interest is the Caulobacterales. Phylotype SL20021 was affiliated with

Nitrobacter sp., a facultative nitrifying chemolithotroph (Zare et al. 2003; published in database only), detected in saturated sediment from Loons but not detected in dry sediment from Loons or Entrance samples. Phylotypes affiliated with Brevundimonas sp. were detected in all moonmilk samples. MON0045 was most closely related to Brevundimonas alba (98.2%), a prosthecate oligotroph (Abraham et al. 1999), and present in particularly high numbers in sample ME2 (-10% of total community). Prosthecae are narrow extensions of the bacterial cell wall containing cytoplasm and it has been proposed that these structures confer a variety of benefits to aerobic heterotrophic bacteria including mechanisms for attachment to solid substrates and enhanced respiration and nutrient uptake (Hedlund et al. 1996). Brevundimonas alba was also present in the DGGE analysis (band DMON2) and isolated from all moonmilk samples, reinforcing its ubiquity in moonmilk.

Members of the Sphingomonadales were detected in sediments and moonmilk samples.

Particularly, phylotype CAV0009 most closely related to Sphingopyxis alaskensis (94.2%) was detected in all samples except for SLl and was detected in DGGE analysis (DMON3). Putatively novel members of the genus Sphingomonas and Sphingopyxis were also isolated from sediments

(SEEOOS) and moonmilk (MAE322). Members of the Sphingomonadales are oligotrophic and found in nutrient limited subsurface environments where they metabolise a large number of aromatic compounds (Fredrickson et al. 1995 Balkwill et al. 1997; Barton et al. 2004). Such metabolic diversity has led to the identification of members of this genus in numerous starved environments including distilled waters and oligotrophic marine ecosystems (Balkwill et al.

96 Chapter 3: Results and Discussion 1997). A novel Porphyrobacter sp. was isolated from moonmilk (MEE338). Members of the

Porphyrobacter are aerobic and photosynthetic bacteria.

Three phylotypes and one isolate clustered within the Rhodobacterales lineage. Two phylotypes from moonmilk (MX10016) and Loons sediment (SL20056) were affiliated with phototrophic Rhodobacter sp. A third phylotype SE10043 from Entrance sediment was loosely affiliated (85.7% sequence similarity) with members of the genera Amaricoccus, isolated from activated sludge. A novel methylotroph from the Rhodobacterales lineage, Paracoccus sp., was isolated from a speleothem in Entrance Cave. Paracoccus sp. can utilise methylamine and methyl formamide (Urakami et al. 1990).

97 Chapter 3: Results and Discussion

.------My.wcoccusjulvus A1233917 L AF3'l20'JI --c======ComaTTWnasEscherCf:h"Wc~f.JMl/~uni lts/osleroni Ml 1224 ..------Riclieltsia pr

Rltodoba.cterales

0.1

Figure 3.5: Phylogenetic dendrogram illustrating the evolutionary relationship between cave taxa and members of the a­ Proteobacteria. The dendrogram was constructed from an alignment of 1000 nucleotide pa;itions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Tltem1oprotei1s te11ax was used as the outgroup species. The scale bar indicates 10% sequence divergence. Colour Code: Black = Clone sequences, Blue = DGGE sequences, Brown = Isolate sequences.

98 Chapter 3: Results and Discussion

~ Proteobacteria

Clones affiliated with the ~-Proteobacteria were the most abundant group detected in this study (102 sequences) and were distributed fairly evenly between sample sites SE2, SLl,

SL2, ME3 and MXl contributing approximately 25-34% to the total diversity sampled (Table 3.2).

In comparison however, no ~-Proteobacteria were detected from sites SEl or ME2.

Phylotypes affiliated with the ~-Proteobacteria are depicted in Figure 3.6, clustering with known chemolithotrophs, particularly hydrogen utilising bacteria, thiosulfate oxidisers, and nitrogen-fixing bacteria. Most phylotypes clustered within the Order Burkholderiales. Several sequences obtained from both sediment and moonrnilk were closely related (94-99.3% sequence similarity) to members of the Commamonadaceae, particularly the Acidovorax group, including the genera Acidovorax, Variovorax, Polaromonas, and Hydrogenophaga. DGGE analysis also detected a member of the Hydrogenophaga in ME3 and MXl (DMON4). A novel Acaligenes sp.

(MEE109) was isolated from moonmilk. Members of the Commamonadaceae and Acaligenaceae are aerobic chemoorganotrophs and some strains are capable of chemolithoautotrophy utilising hydrogen as an energy source. Nitrogen-fixation has been reported for some genera, eg.

Burkholderia, Derxia and Hydrogenophaga (Willems et al. 1991). Phylotypes from sediment and moonmilk were distantly related to members of thiosulfate oxidising genera Thiobacillus,

Limnobacter, Ottowia and Delftia (eg. Spring et al. 2001). A number of clones from moonmilk samples were distributed within five phylotypes affiliated with the Oxalobacteraceae, showing close relationships (>96%) with the genera fanthinobacter, Massilia, Duganella, Oxalobacter and

Herbaspirillum. A number of members of the Oxalobacter group are nitrogen-fixing bacteria associated with plants (Valverde et al. 2003). Members of one genus Duganella are also reported to have chitinolytic properties, most likely associated with the breakdown of organic matter. The

Oxalobacteriaceae appear to be a dominant component of the true calcite moonmilk microbial communities sampled accounting for 24% and 18% of samples ME3 and MXl, respectively.

Further evidence of this is the presence of DGGE band DMON6 clustering withfanthinobacter phylotypes.

99 Chapter 3: Results and Discussion Phylotypes affiliated with the Methylophilales dominated the sediment samples, particularly the saturated sediments from Entrance and Loons Cave. SE20011, most closely related to Methylophilus freyburgensis (93.3% sequence similarity) accounts for 13% of the sampled microbial community in saturated sediment from Entrance Cave. Members of the

Methylophilus genus are methanol utilising. SL00038 most closely related to Methylovorus mays

(94.3% sequence similarity) accounts for 18% of the observed microbial community in saturated sediment from Loons Cave. Members of the Methylovorus are aerobic obligate methylotrophs associated with plants (Doronina et al. 2000). A single phylotype (SL20020) from saturated Loons sediment grouped with the ammonia-oxidising species Nitrosospira briensis (90.2%).

c5- Proteobacteria

Clones affiliated with the o-Proteobacteria were detected in all samples. This phylum encompasses sulfate- and sulfide-reducers that are morphologically diverse and obligate anaerobes. Six clones were distributed among three phylotypes (Table 3.2), thus the o­

Proteobacteria were a minor, though ubiquitous component of the microbial communities sampled. Two types of sulfate-reducers are recognised, those species that reduce sulfate to

hydrogen sulfide (H2S) (eg. Desulfovibrio, Desulfomonas, Desulfotomaculum, Desulfobulbus) and those that reduce sulfate to sulfide (eg. Desulfobacter, Desulfococcus, Desulfosarcina, Desulfonema).

Two phylotypes formed separate deep branching lineages within the Desulfuromonadales (Figure

3.7). MON0018 was detected in all moonmilk samples and represents a putatively novel lineage forming a monophyletic clade with the genus Desulfuromonas (90% sequence similarity to

Desulfuromonas thiophila). Members of this genus are obligate sulfate-reducers and widespread in terrestrial and aquatic environments that become anoxic as a result of microbial decomposition processes (Finster et al. 1997). Phylotype SED0098 present in sediment samples SE2, SLl and SL2 were affiliated with sulfur- and iron- reducing members of the Geobacteraceae. A third phylotype

SE10043 detected in sample SEl, formed a deep branching lineage within the Desulfoarculaceae.

The closest cultured relative to this clone was nitrite-oxidiser Nitrospina gracilis.

100 Chapter 3: Results and Discussion

a ,.------Rickettsia prowazekii M21789 .______Campylobacter jejw1i AF372091 E y ..------Escherichia coli X80725 Nilrosospira brie11Sis AY123800 Nitrosomonadales SL20020 Meth.vlophilusfreyb11rf1.e11Sis AJ517772 Methy/ophi/11s 111ethylotrop/u1s Ll5475 Methylophi/11s /eisi11geri AF250333 Methylophilales SL0008 .----SL0038 Methvlovor11s mays AY486132 Burkholderia sordidico/a AF512827 · SL10008 Burkholderaceae .------SL10014

Der.J.ia g11111111osaAB08948l ME20024 Acali!1,e11es faecalis D88008 Acaligenaceae Acalige11es sp. 2-6 A Y296717 CAVI109 Bordetella pertussis AF366576 Achro111obacter xvlosoxida11s AF510042 D11ga11ella violace~s11iger A Y376163 CAV0006 ME30010 Oxalobacter formige11es U49758

Oxalobacteraceae Ja11thi11obacter a.~aricida1111ws11111 Y08845 DMONS CAVOOOS Oxalobacter for111i11e11es U49758 CAV0021 Herbaspirillw11 frisi11. ~e11se AJ238358 Stero/ibacteriw11 de11itrifica11s strain Chol-ls AJ306683 Co111a111011as testosteroni Ml 1224 Delftia tsur11hate11sis A Y302438 MX10008

Acidovorax valeria11e/lae AJ431731

Po/aro111011as vacuolata U14585 Glacier bacterium FJS31 AY315178 Co11u11amo11adaceae MONOOlO

Hydro11e11opha .~a defl11vii AJ585993 Arsenite-oxidising bacterium AY027499 CAV0004 DMON4

Ottowia thioo.J.vdans AJ537466 SE20028 Thiobacillus de11itrifica11s AJ243144 SL10010 Hydroge11ophilales 0.1

Figure 3.6: Phylogenetic dendrogram illustrating the evolutionary relationship between cave taxa and members of the {3- Proteobacteria. The dendrogram was constructed from an alignment of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Thennoproteus te11a.x was used as the outgroup species. The scale bar indicates 10% sequence divergence. Colour Code: Black =Clone sequences, Blue = DGGE sequences, Brown = Isolate sequences.

101 Chapter 3: Results and Discussion

...------Thermoproleustena.t:r-..f35966

...------Esc11cncl11a coll X80725 y ~------Comamo11as le.stoJ,1erom M11224 ~ a ...------Ru.kettsmprcrn-a::.eku M21789

...------Campvlolxu:ter Jf!JU!lt AF372091 Desulfurel/a/cs '------Desulfurella aceln1oraus X72768

...------Bdellov1bno bactenovoms M59297 Bdellowbrw11ales

..------MJ\OCOCCllSfufrus A1233917 1\fy}.OCOCCales

...------N11rospma gracrlls 1.35504

'------SL0043

...------Desulfobacler postgaler AF418180

Umdenb.fied su]fate reducmg bactenum DSB-Dsa99-4 AJ300510 Desulfobacteralcs

Desulfo11ema 11m1cola U45990

Desulfococcus mulllvorans AF418173

Desulfococws bwt1wtus i\J217'8ff7

Desulfuramonas m.J?lotuiafls l\12~

Dr.sulfuromona.s palm1talls U28172

Desulfuromonas tluoplula Y11560 Desulfaromonadales

.______MON0018

Pelobacter mas11el1enszs A Y 187308

Pelobacteracrd1galhc1 s.tram MaGa12-T X77216

SE10098

Geobacter peloplnl11s U96918

Geobaclc>r h111rureduce11s AY187306

...----- Desulfomomle uedpn :M26635

Sw10tropliobac1erv,,o/11nr X10905 Syntrophobactera/ es

Dcsulfurolwbdus ammgemts X83274

01

Figure 3.7: Phylogenetic deudrogram tllustratmg the evolutionary relauonsh1p betwe.en cave ta"Xa and members of the 0-Proteobactena The dendrogram was constructed from an ahgnment of 1000 nucleotide positions Distances were calculated m DNADIST and trees \\ere inferred h} the ne1ghbour-101mng method Thermoproteus te11cu was used as the outgroup species The scale bar mdicates 10% sequence divergence

r-Proteobacteria

A total of 77 clones in 22 phylotypes were affiliated with the y-Proteobacteria. Figure 3.8 illustrates the phylogenetic distribution of y-phylotypes. The y-Proteobacteria dominated

Entrance sediments SEl and SE2 representing 29.6% and 26.2%, respectively, of the diversity sampled and also represented a significant component of sample SLl (21.8%) (Table 3.2). Several sequences from Entrance sediment SEl clustered within the Order Chromatiales, whose members

102 Chapter 3: Results and Discussion are predominantly phototrophic and includes sulfur-, H 2S- and thiosulfate- and nitrite-oxidising autotrophic genera Nitrosococcus, Thioalkalivibrio, Thioploca, Beggiatoa. Some cultured

representatives are capable of utilising atmospheric C02 as a carbon source for growth in dark conditions. SEl clones affiliated with the Chromatiales represent 19% of total diversity sampled thus inferring that these are a dominant component of the community. SE10058, consisting of six clones, was affiliated with Thioalkalivibrio thiocyanodenitrificans (92% sequence similarity) an obligate sulfur-oxidising/ nitrifying chemolithoautotroph. Two phylotypes distantly related to autotrophic denitrifyer species Nitrosococcus oceani (90-91 % sequence similarity) were detected in both Loons and Entrance sediment. Phylotype SEDOOlO, also detected in both Entrance and

Loons sediment clustered with Thiocapsa roseoperscina, a thiosulfate-oxidiser.

The Pseudomonads (Pseudomonadales and Xanthomonadales) are a diverse group of aerobic chemoheterotrophs that never show fermentative metabolism. Some members are

chemolithotrophic using H 2 and CO as sole electron donors and some members can use nitrate as an electron donor. Within the Pseudomonadales, a number of sequences, distributed in four phylotypes, from sediments and moonmilk clustered with the genus Pseudomonas, most closely related to members of the fluorescent sub-group (P. fluorescens, P.putida, and P.aeruginosa) and a single phylotype from moonmilk clustered with Acinetobacter. Pseudomonads have simple nutritional requirements, the most striking feature being a versatile metabolic lifestyle and the ability to metabolise a range of substrates including numerous aromatic compounds as the sole carbon and energy source.

Several clones were distributed amongst five phylotypes showing high sequence similarity (95-97%) with denitrifying genera of the Xanthomonadales, (Lysobacter, Luteimonas,

Frauteria, Hydrogenocarboniphaga, Pseudoxanthomonas) and a novel Xanthomonas sp. was isolated from moonmilk. Xanthomonadales are also ecologically important in soil and water and are probably responsible for degradation of many soluble compounds derived from the breakdown of plant and animal materials in oxic environments (eg. Lysobacter sp. can lyse both bacteria and fungi through array of lytic enzymes). A second novel Xanthomonad was isolated from moonmilk, the closest cultured relative being Stenotrophomonas maltophilia, which is also the

103 Chapter 3: Results and Discussion closest relative of clones of novel iron-oxidising bacteria (Emerson & Moyer, 1997). Rice et al.

(1995) also found that S.maltophilia studied in biofilms showed exceptionally adhesive and corrosive properties.

Phylotype SED0008 from sediment samples SE2, SLl and 2, clustered with the Enteric bacteria, a homogenous, facultatively aerobic, group within y-Proteobacteria. This phylotype was numerically significant in that it contained nine clones from SE2 and SLl, and 2 clones from

SL2. Phylogenetically, it was most closely related to both Photorhabdus luminescens and

Escherichia coli strain 5.1. P.luminescens is a symbiotic bacteria and E.coli is able to grow on a wide variety of carbon and energy sources.

Other minor components of the y-Proteobacteria clones include, a phylotype (SE20006) closely related to Achromatium oxaliferum (93%) a sulfur-oxidiser that has sulfur and calcite inclusions within the cell, detected in Entrance sediment. Phylotypes, SE10021 and ME30011, were distantly affiliated (90-91 %) with Methylococcus capsulatus, a methane dependant bacteria.

SE10004 was closely related to Acidothiobacillus ferroxidans (98%) a ubiquitously distributed chemolithotroph that derives energy from reduced sulfur compounds or by oxidising ferrous iron to ferric iron (Kelly & Wood, 2000). Aferroxidans is also capable of autotrophic growth by

C02 fixation. No y-Proteobacteria clones were detected in sample ME2 or in DGGE analysis.

104 Chapter 3: Results and Discussion

Rickettsia prowazekii M21789 a ..------Campylobacterjejuni AF372091 e Myxococcus f11/vus AJ233917 a .------Co111amo11as testosteroni Ml 1224 y '------Dichelobacter 11odosr1s M35016 .-----SE10004 ._____ Acidithiobacillus ferrooxidalls AJ457808 AcidithiobacilJales SE1004S Lysobacter g11111111os11S AB161361 Ste11otrooho111011as 11ilritireduce11s Ste11otropho111011as maltophi/a X95923 CAVIUO Xanthomonadales

Fra111eria a11ra111ia AJ010481 SLlOOSl Ni1rosococc11s halophi/11s AJ298748 Nilrosococc11s occa11i AF363287 SED0007 Chromatiales SL10022 .---- Aero111011as 1110/111scon1111 A Y 532692 .------Vibrio cltolerae X76337 .------Pasteurella 11111/tocida Escheric/1ia co/i X80725 Esc/1eric/1ia co/i str.5.2 AY319393 SED0008 Enterobacterales P/10/orhalxius /11111i11esce11s 078005 Silieella fle.rncri X96963 ...--- - Ac/1ro111ati11111 oxalifen1111 L48227 '------SE20006 Legio11e//a /011di11ie11sis Z49728 SEHl003 Le11io11el/ales Me1hvlococc11s caps11lat11s X72770 '-----L_ __Jr-- ME30011 Metftylococcales SE10021 ..---- Nitrococcus mobilis 135510 Alkalispiril/11111 mobile AF114783 SEDOOlO

Thiobacil/11s prospems AY034139 Chromatiales Th ioalcalovibrio 11itra1us AF126547 Uncultured bacteriwn clone Cll-Kll Thioalkalivibrio thiocva1uxiP11i1rifica11s AY360060

Pse11do111011as f11wresce11s AF094729 SE20012 Pse11do111oll(IS p111ida AF094743 Pseudo111011adales SE20013 Pser.1do111011as a11g11i/lisep1ica X99540 Mari11obac1eri11111 georgiense AB021408 SED0012

0.1

Figure 3.8: Phylogenetic dendrogram illustrating the evolutionary relationship between cave taxa aud members of the y­ Proteobacteria. The dendrogram was constructed from au aligmnent of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Themwproteus te11ax was used as the outgroup species. The scale bar indicates 10% sequence divergence. Colour Code: Black = Clone sequences, Brown = Isolate sequences.

105 Chapter 3: Results and Discussion Actinobacteria

The Actinobacteria were the second most commonly sampled group overall behind the 13-

Proteobacteria though not always the second most abundant group in individual libraries.

Unlike the 13-Proteobacteria, phylotypes affiliated with the Actinobacteria were detected in all sediment and moonmilk samples. A total of 85 clones were distributed among 37 phylotypes illustrating the broad diversity of Actinobacteria sampled in this study (Table 3.2). Particularly, the Actinobacteria were the second most abundant group in sediment sample SEl and mat sample ME2, both from Entrance Cave, composing 36.6% and 26.8%, respectively, of the total sampled clonal diversity. DGGE analysis revealed two Actinobacteria taxa in moonmilk samples,

DMON6 and DMON7 (Table 3.2). Isolations from sediments, speleothems and moonmilk samples were dominated by Actinobacteria resulting in cultured representatives from 14 genera, including one putatively novel genus and five putatively novel species (Table 3.3).

The Pseudonocardineae dominated the clone libraries and revealed great diversity. A total of ten phylotypes were detected (Figure 3.9) and were particularly abundant in calcite sample

ME2 with 6 phylotypes consisting of 15 clones. Several sequences from sediment and moonmilk were affiliated with the genus Saccharothrix most closely related to various described species.

17% of the total diversity sampled in ME2 were affiliated with Saccharothrix species illustrating the dominance of this taxa in the calcite samples. DGGE analysis also revealed the presence of

Saccharothrix sp. in calcite moonmilk samples (DMON5). Saccharothrix sp. were isolated from moonmilk and sediment, including S.albidocapillate, S.cryophilus and S.violacea. S. violacea is a chemoorganotrophic strict aerobe that was isolated from soils inside a gold mine cave in Korea

(Lee et al. 2000) and has been detected in other caves (Schabereiter-Gurtner et al. 2002, 2004;

Northup et al. 2003). A novel Amycolatopsis sp. was isolated from sediment from Entrance Cave.

Clones and isolates affiliated with the genera Micromonospora, Couchioplanes and Actinoplanes were also present from sediments and moonmilk. Sequences clustering within the

Propionibacterineae were detected in sediment and moonmilk samples (Figure 3.9). A phylotype closely related to Propionibacterium acnes (98%), a common human skin commensal, is probably a contaminant. Clones related to Nocardioides fulvus (94% sequence similarity) and Pimelobacter

106 Chapter 3: Results and Discussion simplex (98%) were detected in sediment and calcite samples. Members of the Nocardioides are oligotrophic and able to support growth on a wide variety of substrates (Yoon et al. 1999). A single sequence only distantly related to Blastococcus saxobsidens (90%) within the Frankineae was detected in Entrance Cave sediment. Several members of the Frankineae including Blastococcus, have been isolated from monuments. Several sequences detected in Entrance sediments were distantly related to the genus Frankia. Frankia sp., are nitrogen-fixing bacteria usually associated with plants.

107 Chapter 3: Results and Discussion

.------Bifidobacterium bifidwn S83624 Nocardioi

,..------1.:.P.:.ro~'P:_:ibnibactuium ac""s AB042288 CAV0023

Virgosporangium ochrace1m1 AB006167 MX10039 Micro11wnospori.11.eae Micromonospora chalcea U58S31 Micronwnaspora echinoauranliaca X92618 SL10009

Actinobispora alani,,iphila AF325726 r----- SL10013 Pseudonocardia spinospora Pseudomxarditteae asaccharolytica

Actinosyniemma. mirum X84447 Pseudo11ocardineae Saccharothrix violacea AJ24Ui34 u1ur.ea a/bidocapi//ata X84321 lenJuajlavoverrucosus AF183957 .------CAVl312

Crossiella equi AF'245017

Saccharothrix cryophilus AFI 14806

,----- SL20046 Kibdelosporangium phillipi11ense AJ512464 r-----SE0098 Franldasp. SE10039 I Frankineae Blasrococcus sa:cobJidens AJ316570 0.1

Flgare 3.8: Phylogenetic dendrogram illustrating the evolutionary relationship between cave taxa and members of the Actinomycetaks. The dendrogram was constructed from an alignment of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Bifidobacteriwn bifidum was used as the outgroup species. The scale bar indicates 10% seqnence divergence. Colour Code: Black ; Clone sequences, Blne ; OOGE sequences. Brown ; Isolate sequences

108 Chapter 3: Results and Discussion

~------Biftdobaeterlum bifidum S83624 Cor:mt!bocteritml diptheriae X84248 Tsulcamurelln pourometabola X80628 Tsukomurella stro11djordo.e AF283283 Tsukamurella pulmonis AY254698 ~--- CAVl306 Rhodococcus rltodoclirous X80624 Rilodccoccus erylh"opolls X80618 CAVI203 R11odococc11s globerulus X80619 CAVl104 CAVl321 Corynebacteri11eae

Nocardia cummideleus AF430052

Agromyces ro111 os11111 X774'f"/ Agromyces aurantiacus AF389342 Brevibacterium linens AF426135 Brevibacterium iodi.Jam1 X76567 CAVl2S Micrococci11eae Arthrobacter oxydtuJS X83408

'----4 CAV0046 Anhrobacter chlorophe110/icus AF102267 CAVIOOS DMONS Kocurin roscus CAVl117 Brachybactcrium paraco11glc111eralum AJ415377 Braclrybactcriru11 fresconis AJ415378 Bracltvbacterium r/lamnosum AJ41S376 CAVJ31S CAV1002 ~---- Micrococcu.s lylae X80750 ME30023

CAVl318 Arthrobacter pasce11s X80740 0.1

Flgarc 3.10: Phylogenetic dendrogram illustrating the evolutionary relationship between cave taxa and members of the Micrococcineae and Cory11ebacteri11eae. The dendrogram was constructed from an alignment of 1000 nucleotide pooitions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Bifidobacterium bifidwn was used as the outgroup species The scale bar indicates 10% sequence divergence. Colour Code: Black =Clone sequences, Blne = DGGE sequences, Brown = Isolate sequences

Figure 3.10 is a phylogenetic dendrogram of the Microccineae and Con;nebacterineae.

These bacteria are among the most common organisms isolated from caves. Several strains of

Arthrobacter were isolated from cave sediment and moorunilk. One group of Arthrobacter moorunilk isolates were related to Arthrobacter chlorophenolicus (96 % similarity). This is a

putatively novel cave species that was also represented in DGGE analysis (DMON4).

Arthrobacter is one of the main genera of Micrococcineae, and consists mainly of soil organisms. 109 Chapter 3: Results and Discussion Arthrobacter sp. are remarkably resistant to desiccation and starvation, despite not forming spores and demonstrate considerable nutritional versatility including the ability to decompose a variety of organic compounds. Members of the Arthrobacter have previously been observed in caves demonstrating survival by means of nitrogen fixation or the use of organic substrates as the sole source of carbon and energy, and remain resistant to prolonged periods of nutrient limitation (Barton et al. 2004). A phylotype very closely related to Knoellia subterranea (99%) was detected in sample ME3 and was also isolated from sample ME2. Knoellia sinensis and Knoellia subterranea, were recently isolated from sediment in Reed Flute Cave in China (Groth et al. 2002).

Two phylotypes affiliated with the genus Nocardia were detected in sample ME2, ME20019 being almost identical to N.carnea (99%) and ME20104 being more distantly related to

N.corynebacteroides (94%) perhaps representing a novel species of the Nocardia. Coryneform bacteria, Nocardia and Rhodococcus, are soil organisms sometimes utilising hydrocarbons. Species of these genera are known to degrade organic matter and are able to decompose environmentally hazardous chemical compounds. Several Nocardia and Rhodococcus sp. were isolated from all sediment and moonmilk samples and although Rhodococcus sp. were not detected in culture-independent analyses. Members of the genus Rhodococcus show a remarkable degree of metabolic diversity and currently are used as whole-cell biocatalysts in several industrial processes (Hughes et al. 1998).

Phylotypes affiliated with the Streptomyces were ubiquitous in cave samples (Figure

3.11). Members of the Streptomyces dominated isolations from sediment and moonmilk accounting for approximately 60% of isolates obtained. These isolates represented 10 species of

Streptomyces (Table 3.3) The most common species isolated were S. subrutilus and S. caviscabies. S. subrutilus was detected in all sediment, speleothem and moonmilk samples and clones clustering with this lineage were detected in Entrance sediment and calcite mat material, ME2. S. caviscabies was isolated from all samples except for ME2. It was also detected in Loons sediment and moonmilk samples. The genus Streptomyces encompasses a large number of recognised species. Streptomyces are the most common soil bacteria along with the Arthrobacter. Members of the Streptomyces favour alkaline to neutral, well drained soils such as sandy loams or soils

110 Chapter 3: Results and Discussion covering limestone. Limestone caves and lava tube caves often contain wonderful displays of filamentous actinomycetes that may cover entire ceilings and walls of caves giving a 'silvered'

appearance (similar to sample ME2). Probably many of the discrete lichen-like colonies frequently noted on walls and formations in the dark zone may be Streptomyces species since they often have the powdery appearance and characteristic earthy odour common to cultures of this genus. Several of the isolates from sediments and moonmilk in this study had this powdery

appearance and earthy odour. It has also been suggested that the abundant Streptomyces in caves is probably responsible for the earthy smell of caving (Caumartin, 1963 in Ford & Cullingford,

1976).

111 Chapter 3: Results and Discussion

~------Bifidobacterium bifidum 583624

Streptomyces violaceon1ber AF503492 Streptomyces gougerotii Z76687 Streptomyces mt~ersensis Z16688 ME20041 CAVl314 SEOOSO ----CAVOOIS Streptomyces macrospoms Z68099

Streplomyces beijiangensis AF385681 Streptomyces aureus AY094368

treptomyces microstreptospora AB006l 59

Streptomyces sanglieri AY094364 CAV1231 CAVIOOS Kitasatospora mediocidica U93324 Kitasatospora setae U93332 SL10055 CAVl317 SL10019

Streptomyces virf(iniae 085121

Streptomyces himgiriensis A Y370772

Streptomyces caviscabies AFI 12160 CAV1025 CAVJ116 CAVIOIO CAV1313 CAVIOl9 CAVIOOI 0.1

Figure 3.11: Phylogenetic dendrogram illustrating the evolutionruy relationship between cave taxa and members of the Streptomycineae. The dendrogram was constructed from an alignment of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbour·joining method. Bifidobacterium hifidum was used as the outgroup species. The scale bar indicates I 0% sequence divergence. Colour Code: Black = Clone sequences, Brown = Isolate sequences.

112 Chapter 3: Results and Discussion Four phylotypes were detected clustering within the Actinobacteria but not affiliated with the

Actinomycetales. Figure 3.12 is a phylogenetic dendrogram of Actinobacteria subclasses

Rubrobacterideae and Sphaerobacterideae and unclassified Actinobacteria. Described members of the

Rubrobacterideae are largely thermophilic (eg. Thermoleophilum minutum, Thermoleophilum album,

Rubrobacter radiotolerans). Two clones, from moonmilk samples, were loosely affiliated with members of the thermophilic genus Thermoleophilum (89-93%) forming a monophyletic radiation within the Rubrobacterideae and perhaps representing cold-adapted members of this taxa. The

Rubrobacterideae are a broad monophyletic group within the Actinobacteria consisting of to date largely uncultivated organisms (Rheims et al. 1996). Culture-independent studies have detected members of this group as ubiquitous and an ecologically significant radiation of the

Actinobacteria, inhabiting a diverse array of environments including peat bog (Rheims et al.

1996), forest soil (Liesack & Stackebrandt, 1992), geothermal soil (Fuhrman et al. 1993), paddy and soybean fields (Ueda et al. 1995) and marine habitats (Fuhrman et al. 1993). Within the unclassified Actinobacteria, 2 clones from Entrance sediment, SEl, were distantly related to

Candidatus Microthrix parvicella (91 %). Microthrix parvicella is a filamentous organism isolated from an activated sewage treatment plant. One sequence, also from SEl, was loosely affiliated with thermophile Sphaerobacter thermophilus.

As confirmed in this study, actinomycetes are the most common and abundant group isolated from caves samples and are detected consistently, though in moderate numbers, in culture-independent studies. Streptomyces species are particularly abundant and in some cases, can be found as apparently monospecific colonies (Arroyo & Arroyo, 1996). A number of actinomycetes isolated from caves have the ability to produce various types of crystals. Studies in Altamira and Tito Bustillo Caves demonstrate that the host-rock (bedrock), cave formations and rock art paintings are coated by dense networks of bacteria, mainly actinomycetes and these bacteria can induce constructive (calcification, crystalline precipitates) and destructive (irregular etching, spiky calcite) fabrics. Because of this ability it has been proposed that these bacteria and others are directly or indirectly involved in constructive biomineralisation processes in caves

(Laiz et al. 1999; Barton et al. 2001; Canaveras et al. 2001; Groth et al. 2001; Jones, 2001). Little is

113 Chapter 3: Results and Discussion known concerning the distribution, population dynamics, growth rates and biogeochernical processes of Actinobacteria in caves, in spite of the fact that they seem to constitute a significant part of the "culturable" microbial population of these habitats. A prerequisite for the study of the role of actinomycetes in biogeochemical processes is the isolation and identification of these organisms (Groth et al. 1999a) .

.------Tliemroproleus te11a.\ J\!35966 SElOOOl SpllfU'robactl'T'uleae

Uncultured bactenwn clone FBP471 AY250886

Conobaccen11111 glomerans X79048 Conobacteruleae

Rubrobactendeae

RI1brolxwer rad101oleraus U656-f.7

Umdennfied bactenum \\bl...1'06 AF31'Tl69

SE200SO SE10037 Femmurolmmi ac1d1pluhm1 AF251436 l Inclassif1ed Ach11obactena Catliayosporangmm alboflavum AB006158

SE10060

Candi.datus ,\>ficrot1mx pmvicellcr X89774

Pse11do11ocardm tl1ermophila AJ252830

Streptomyce" allmi; X53163

Micromonospora cllf'llcea U58531

Fronkto. sp AF034716

Gfycomyce.s lwrlnnensis AJ293747

Acul1m1crobu11nferrooJ.1da11s U7%47 Actmobactendeae

Btfidabaclenum b1fidum S83624

Aclmo111)ce~ buv1sX81061

Micrococcus lylae X80750

Corynebaclena d1pthen.ne X842..J8

Propiombactanum acnes .<\.B042288 __0_1_

Figure 3.12: Phylogeneuc dendrogram tllustratmg the e\•olutmnmy relattonshtp between cave ta'l(a and members of the 4.rtmolwNendeae The dendrograrn was constructed from an ahgnment of 1000 nucleotide pos1tmn'i DIStances were calculated m DNADIST and trees "'ere mferred by the ne1gbbour-J01mng method Thennoproteus tena.x was used as the outgroup species The scale bar md1cates 10% sequence divergence

114 Chapter 3: Results and Discussion Finnicutes

Few clones in this study were affiliated with the Finnicutes (low G+C Gram-positive bacteria). A total of five sequences distributed in three phylotypes were detected (Table 3.2). In contrast, 17 moorunilk isolates distributed across seven strains were identified as members of the genera Bacillus, Paenibacillus and Sporosarcina. Figure 3.13 illustrates the phylogenetic relationships of phylotypes and cave isolates to cultivated members of the Firmicutes. Two phylotypes were detected in moomnilk samples. MX10063 was closely related (99%) to Bacillus subtilus, also isolated from samples ME3 and MXl. Remaining Bacillus species isolated include B. simplex, B. pumilus, B. indicus, and B. mycoides, cultured from all moomnilk samples (Figure 3.13).

Bacillus sp. are aerobic, endospore forming and mainly found in soil. ME20098 was affiliated with Sporosarcina ureae and strains of this microbe were isolated from sample ME2 and ME3.

Members of the genus Sporosarcina are strictly aerobic. S. ureae is common in soils with urea input and is perhaps an important ecological degrader of urea. A single phylotype affiliated with Finnicutes was detected in sediment, SL10009, showing a distant relationship (91%) to

Ruminococcus flavescians, usually detected as a symbiont in the gut of animals.

115 Chapter 3: Results and Discussion

Ther111oprote11s 1e11a.x !Vf35966

.----- lactobaci/lus delbmeckii M58814 Streptococcus cristalus AY281090 Lactobacillales Streptococcus 1hora/te11sis C/ostridium butyricum AB07S768 Clostridia ~------Mycop/asma mycoides M23943 Mollicutes ~----- Rumi11ococcus jlavescie11s XS.5091 ...------Anaerobic bacterium LR7.2 A Y327251 .....______SL10009 Clostridia

Pae11ibacillus polymyxa AJ320493 Pae11ibacillus turice11sis clone B2 AF378699 Pae11ibaci l/11s W\/llllii AJ633647 Pae11ibacillaceae Pae11ibacillus graminis strain RSA19 CAVI309 Geobacillus stearother111ophi/11s strain R-20093 AJ586387 Pla11ococcus cilrea X62172

Sporosarcina 111ac11mrdoe11sis CMS 21 w AJ514408 Permafrost bacteriumDT-ID02 AY378272 Pla11ococcaceae Filibacter limicola AJ292316

Bacil/11s coh 11ii X76437 Bacil/11s /iche11ifor111is X60623 Bacillus pumilus AY456263 CAVl102

Bacillaceae

0.1

Figure 3.13: Phylogenetic dendrogtam illustrating the evolutionary relationship between cave taxa and members of the Fimticutes. The dendrogtam was constructed from an alignment of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbout-joining method. Themwproteus te11ax was used as the outgroup species. The scale bar indicates 10% sequence divergence. Colour Code: Black = Clone sequences. Brown = Isolate sequences.

116 Chapter 3: Results and Discussion

....------Then11oproteus tenaxM35966 .------Chlorobium limicola AJ290824 Chlorobi

Bacteroidales

.----- SLI0019 Flexibacter tructuosus M.58789

Sphi1111obacteriw11 multivora11 014025 Sphi1111obacteriw11 faeciuJn AJ438176 Sohineobacterium soirilivorw11 014026 Sphingobacteriales Glacier bacterium FJSS AY315161 Uncultured Bacteroidetes bacterium clone Bisi29 AJ318173 CAVI317

Pedobacter heparinus AJ438172 ...------Cryotnorpha ignava AF170738 .----- Arctic sea ice bacteriumARK10177 AF468426 MX10045 ~----- SL10020 Flavobacteria columnare M58781 FlavobacteriuJ11 aquatile M62797 Flc111obacteria ferruginew11 M62798 SL10003 Flat>obacteila limicola AB075230 Flavobacteriales CAVOOlS

CAVOOJO Antarctic bacterium R-7933 AJ440987 Bacterium C:Sl 12 AYl24338 C AVI311 F/ll!•obacteria leeana AB180738 Glacier bacterium FJS20 AY315160 Elbe River snow isolate Iso8 AF150713 SL10035 CAV0018 DMON6 0 I

Figure 3.14: Phylogenetic dendrogram illustrating the evolutionaiy relationship between cave taxa and members of the Cytoplraga-Flexi.bacter-Bacteroides group. The dendrogram was constructed from an alignment of 1000 nucleotide positions. Distances were calculated in DNADIST and trees were inferred by the neighbour-joining method. Then11oproteus tenax was used as the outgroup species. The scale bar indicates 10% sequence divergence. Colour Code: Black= Clone sequences. Blue = DGGE sequences. Brown = Isolate sequences.

CFB Group

A total of 65 clones distributed in 14 phylotypes were affiliated with the CFBs (Table

3.2). The CFBs were the second most abundant major phyla detected in moonm.ilk samples ME3 and MX1. Several of these sequences clustered with psychrophilic Flavobacteriaceae (Figure 3.14) that are represented by various aerobic and heterotrophic genera. Several sequences from both samples ME3 and MX1 were closely related to Flavobacteria limicola (98%) a psychrophilic, organic polymer degrader (Tama.ki et al. 2003). This phylotype was present in DGGE analysis

117 Chapter 3: Results and Discussion and represented 10-12% of the total diversity sampled in the moonmilk clone libraries, demonstrating its dominance in these habitats. A novel Flavobacteria sp. was isolated from moonmilk sample ME3 clustering with the F. leeana-Iike sequences. A single sequence MX10045 showed distant similarity (92%) to Cryomorpha ignava a cold-adapted, strict aerobe isolated from

Antarctic quartz stone subliths (Bowman et al. 2003). Phylotypes ME30011 and MON0015 from moonmilk clustered with psychrophilic members of the Sphingobacteriales, genera

Sphingobacterium and Pedobacter (Figure 3.14). Phylotype ME30011 represented by three clones was related to Pedobacter cryconitis, a facultative psychrophile isolated from an alpine glacier

(Margesin et al. 2003). Phylotype MON0015 was affiliated with Sphingobacterium faecium. A number of uncultured glacier and sub-glacial sediment clones (FJS and FX clone groups) clustered with moonmilk phylotypes identified in this study, inferring the presence of cold­ adapted taxa in these samples.

Three phylotypes clustered within the Bacteroides group. SL0019 branched with

Flexibacter tructuosus (92% sequence similarity). Phylotype CAV0026, detected in sediment and moonmilk samples from Entrance Cave, is distantly related to uncultured Bacteroides bacterium

Mo-0.2plat-K3, detected in freshwater. Phylotype SL10028 was not closely affiliated with any described taxa, however it clustered with a group of uncultured bacterial clones from

Palaeolithic rock art in Spanish and Italian caves within the Bacteroides clade (Figure 3.14). The

Bacteroides group includes a mixture of physiological types such as strictly anaerobic Bacteroides and aerobic gliding bacteriCJ. such as Flexibacter. Bacteria with gliding motility have no flagella but are able to move when in contact with surfaces.

Acidobacteria

A total of 11 clones affiliated with the Acidobacteria were detected in the cave samples.

Most clones form a monophyletic clade within sub-Phylum A of the Acidobacteria showing varying degrees of similarity to uncultured bacterium DA008 (90-94%), a clone from grassland soils (Figure 3.15). These sequences were retrieved from moonmilk samples, particularly sample

ME2 (5 clones). The remaining sequences were affiliated with Ellin isolates from Australian soils

118 Chapter 3: Results and Discussion (Sait et al. 2002; Joseph et al. 2003) within sub-Phyla C and D. Though the Ellin group represents cultured members, these have not been described to date thus no information is available about their physiology or metabolism. The Acidobacteria are a relatively cosmopolitan group, widely distributed in the environment though in general are highly correlated with the soil habitat.

(Hugenholtz et al. 1998). The division was defined by Ludwig et al. (1997) on the basis of cloned

165 sequences from soil, freshwater sediments and activated sludge in many geographic locations and its members are thought to be ecologically significant in many ecosystems.

However it is a poorly studied division thus far, consisting of only a few cultured representatives: Acidobacterium capsulatum an acidophilic chemoorganotroph from acid mineral environment (Kishimoto et al. 1991), Geothrix fermentans an iron-reducing bacteria from a hydrocarbon contaminated aquifer (Coates, 1999), and Holophaga foetida a homoacetogenic bacterium degrading methoxylated aromatic compounds (Liesack et al. 1994).

119 Chapter 3: Results and Discussion r------Then11oproteuste11a.\1-135966

Bactenal speaes (clone 11-14) Z95710

'----- SL10020 Sub-Phyla D 4.c1dolXlcler11m1 capsulalum D26171

Bactenwn Ellm5289 AY23-l6-IO

Bactenwu Bhn6CT75 AY234727

ME20020 Sub-PhylaC

Uncultured bactenum clone Alt9-h."71 AJ421902

Uncultured bactenum cloneC11-K25AJ421117

SL20005

~---- Uncultured bactenum clone Alt9-K74 AJ..J.21904

Bactenum Elhn6100 AY234752

Baclenmn Elhn6071 AY234723

Geot/lru .frrme11ta11s U41563

Holophagafoeluia X77215 Sub-Phyla B

'------Uncultured bactenwn clone C'4-K19 AJ421210

llncultured bac1emnn DA008Y112597

Sub-Phyla A MONOIJ.J5

Uncultured bactemun clone C2-K16 AJ421198

ME20013 01

Figure 3.15': Phylogenetic dendrogram 1llustratmg the evoluuonary relat10nsh1p between cave ta\.a and members of the Ac1dobacterza The dendrogram was con;tructed from an alignment of 1000 nncleoude posmons Distances were calculated m DNADIST and trees were mferred by the ne1ghlxmr-Jommg method Themioproteus temv.. \\"1\S used as the outgroup species The scale bar md1cates 10% sequence dn ergence

120 Chapter 3: Results and Discussion Planctomycetales

A total of 31 clones affiliated with the Planctomycetales were detected in sediment and moorunilk samples (Figure 3.16). The majority of these clones belonged to six deeply branching phylotypes within the genera Planctomyces, Pirellula and Gemmata, showing distant (87.2-90% similarity) relationships to cultured members. This is not suprising as the intralineage phylogenetic depth of the Planctomycetales was recently expanded to 30.6% (Chouari et al. 2003).

One phylotype (SED0047) detected in all sediments, was closely related to Pirellula staleyi (96%).

Four cultured genera, consisting of seven species overall, have been described to date,

Planctomyces, Pirellula, Gemmata and Isophaera (eg. Schlesner, 1986; Giovannoni et al. 1987;

Schlesner 1989). All these organisms are aerobic chemoheterotrophs. Knowledge of this group is limited because of the relatively few species that have been obtained in pure culture.

Membership of the planctomycete group has been extended not only to chemoorganotrophs and obligate or facultative aerobes but also to obligate anaerobes, autotrophs and phototrophs, demonstrating diverse metabolic properties within this line of descent (Fuerst, 1995; Miskin et al.

1999). For example, a planctomycete was found to be responsible for anaerobic oxidation of ammonia (Strous et al. 1999). All Planctomycetales were originally isolated from aquatic habitats as diverse as acid bogs and sewage treatment plants though culture-independent studies have revealed the presence of Planctomycetales in more diverse environments including marine, sediment, anoxic bioreactors, anoxic sediments and caves (DeLong et al. 1993; Godon et al. 1997;

Holmes et al. 2001; Tay et al. 2001; Chouari et al. 2003). The Planctomycetales were a significant component of Entrance Cave dry sediment being the third most abundant group detected in sample SEl (22.5%) whereas in all other samples they constituted a relatively minor component of the community (1-8%).

121 Chapter 3: Results and Discussion

..------T1wmzoproteus tmaxM35966 -----Pla11ctomvccs bras1lie11srs AJ231190 '-----Planctom:yces marts AJ2.o1184 ~---SED0062 '----sE10039 ~---- Pirellu/a sta/eyz AJ23 WB

Planctomycetales Uncultured scnl bactenum PRR-7 A T390478 Uncultured soil bactenum PRR-47 AJ390484 Uncultured s01l bactemlll.l PRR-8 A..1390-1-79 r------Bactenum (s01l clone MCl 1) X64378

'-----Bactermm DR2A-7Gl 9 AB127858 ....------ME.10013 Genuriata-hke str. Cjuq14 AF239693 ~--- Gemmata obmmglobus AJ231191 ,------CblamvdmtrachonUJhs stram H..\R-13 089067 ..------Prost.hecobacler fusifonms U60015 '------Unculnrred verrucomicroblllm DEV003 AJ401104 .------Verrucom1crobia spmosum X90515 Uncultured ;erruconncroblllm DEVlO AJ401127 ~--- SE10094 Verrucom1crohia . SEI0006 Omtutus terrae stram PB90-1 AJ229235 Op1tutus sp YeSm13 X99392 ~------f"------Clllorojle.A.11smuantzacus AJ308500 ~------Green non-snlfur bacternun AK-6 AB079644 ..------Uncultured bactenum clone B1111l'.:? AJ3!815'.:? SE10094 ...._ __ _, MX10044 l\IE20011 ~----- Dehalococco1des ethenogenes AF004982 ~----- Unculnued eubactenum WCHBl-80 AFOS0563 ..----- Cald1lznea aerop/11/a AB067647 Chlorojlexi ~----- Uncultured bactemun S TA-15 41009453 ------ME30006 ..---- Urudeutlfied bactenum stram BD3-16 AB015556 l\

Figure 3.16: Phylogenenc dendrogram tllustratmg the evolutmnary relauonslnp between cave ta"'

Chloroflexi (green non-sulfur bacteria)

Clones affiliated with the Chloroflexi (green non-sulfur) bacteria were detected in

Entrance Cave sediments and moonrnilk samples (Figure 3.16). Members of the Chloroflexi are filamentous and exhibit gliding motility. Chloroflexus, though a phototroph, can grow chemoorganotrophically in the dark under aerobic conditions. Many members of this group digest cellulose or chitin and are widespread in soil and water. Two phylotypes detected in moonrnilk samples (ME30006 and MX10041) were deeply branched within an, until recently,

122 Chapter 3: Results and Discussion uncultivated lineage of Chloroflexi bacteria, sub-phyla I. Sekiguchi et al. (2003) described

Anaerolinea thermophila and Caldilinea aerophila, thin filamentous thermophilic microbes found in abundance in methanogenic granular sludges, representing this lineage. Sub-phyla I is the most diverse of divisions in the Chlorofiexi with sequences derived from hot springs, subsurface environments, aerobic and anaerobic waste water treatment sludges and contaminated aquifers, which hints at its ecological and physiological breadth (Chandler et al. 1998; Hugenholtz et al.

1998; Sekiguchi et al. 2003). A novel lineage represented by two phylotypes detected in moonmilk samples (ME20011 and MX10044) forms a monophyletic clade with sub-phyla II representatives. Dehalococcoides ethenogenes an anaerobe is?lated from activated sludge, able to reductively dechlorinate tetrachloroethane, a common contaminant of groundwater is the most closely related cultured representative of this group (Maymo-Gatell et al. 1999).

Verrucomicrobia

Two sequences from the Entrance Cave dry sediment sample (SE10094 and SE10006) were phylogenetically affiliated with the Verrucomicrobia (Figure 3.16). The division

Verrucomicrobia contains very few cultured representatives but a large number and diverse range of clones from extremely diverse environments including forest soil (Liesack & Stackebrandt,

1992), soybean and rice paddy fields (Ueda et al. 1995); lake (Hiorns et al. 1997); and marine

(Fuhrman et al. 1993) environments. This diversity prompted Hugenholtz et al. (1998) to declare they represent a ubiquitous branch of the domain Bacteria. SE10094 was most closely related to uncultured verrucomicrobium clone DEVlO (92% sequence similarity). The only cultured member of this lineage is Verrucomicrobium spinosum (86.5% sequence similarity to SL10094), an aerobic oligotrophic and chemoheterotrophic prosthecate bacteria (Staley, 1968). SE10006 was phylogenetically affiliated with a relatively newly described genus of the Verrucomicrobia,

Opitutus (Chin et al. 2001). SE10006 was most closely related to Opitutus sp. VeSm13 (91.8%) a novel obligately anaerobic ultramicrobium isolated from anoxic rice paddy soil Ganssen et al.

1997).

123 Chapter 3: Results and Discussion The Verrucomicrobia are often a numerically ~bundant component of soil microbial communities; Buckley & Schmidt (2001) found that the Verrucomicrobia contributed -1.9 % of diversity in 85 soil samples analysed. However no clones affiliated with Verrucomicrobia were detected in sediment samples SE2, SLl or SL2, or any moonmilk samples. Statistically significant variations in verrucomicrobial rRNA gene abundance can be explained by changes in soil moisture content (Buckley & Schmidt, 2001), perhaps explaining the absence of Verrucomicrobia in the more saturated sediments and moonmilk samples in this study.

OP10

Phylotypes SL20004 and SL20017 were affiliated with Candidate division OPlO (Figure

3.17). Clone analysis of sediments from Opal Pool, a hot spring in Yellowstone National Park, yielded representatives of 12 novel lineages designated the OP 1-12 Candidate Divisions

(Hugenholtz, 1998). The Loons Caves phylotypes are deep branching representing putatively novel lineages. SL20004 is most closely related to uncultured bacterium SJA-176 (86%) and

SL20017 is most closely related to uncultured bacterium GC55 (90.2%) detected from a full-scale activated sludge plant (Dalevi et al. 2001). Candidate Division OPlO consists entirely of environmental sequence data with no reported cultivated members to date thus nothing is known of their metabolic or physiological activities. OPlO phylotypes have been detected in hydrocarbon contaminated soil suggesting that this lineage may represent an ecologically significant group (Hugenholtz et al. 1998).

Gemmatimonadetes

Two phylotypes, SL20096 (three clones) and SL20036, detected in Loons Cave sediment samples were phylogenetically affiliated with the Gemmatimonadetes (Figure 3.17). The

Gemmatimonadetes is a new phylum consisting of one described species, Gemmatimonas aurantiaca

(Zhang et al. 2003) and numerous environmental sequences. G. aurantiaca is a Gram-negative aerobic polyphosphate-accumulating microbe. Despite there being only one described species, recently Joseph et al. (2003) were able to isolate a number of Gemmatimonadete species from

124 Chapter 3: Results and Discussion Australian soils (Ellin isolates). Prior to the description of G. aurantiaca the environmental clones in this phylum were designated the candidate division BD and have been found in soils and activated sludge (Hugenholtz et al. 2001), deep sea sediments (Li et al. 1999) and Antarctic sediment (Bowman & McCuaig, 2003). SL10036 and SL20033 were only distantly related to G. aurantiaca (86.5-87.5% sequence similarity) and formed a deeply branching monophyletic clade within the Gemmatamonadetes most likely representing a new group. This low sequence identity is not uncommon in this phylum. Environmental sequence data suggests that members of this phylum are widespread in nature and have a phylogenetic breadth (19% 16S rRNA gene sequence divergence) that is greater than well-known phyla such as the Actinobacteria (18% divergence) (Zhang et al. 2003).

,...------'Dlllmopmteusf-1,.•1uu 1.135966

.------Uncultured lnctenumSJA-22 AJ009456

~----SL20004

Uncultured 1.:nctermm SJA-17 AJ009504 Candidate

~-- UncultureclbactennmGC55AJ271048 D1v1sion OPlO

.....______SL20017

Uncultured 00.ctermm SJA-121 AJ009490

Uncultwed bactenum SJA-176 A.1009504

~------Canchdate dr.is1011 OPlO clone OPB50 AF027092

.------Uncultured Gemmatononas sp cloneBolB6 AB072735

Gemmatimonadetes

Bactenuw Ellm5290 AY2.34641

Bacterium Ellm5220 AY234571 01

Figure 317• Ph}logenetic dendrogram 11lustratmg theevohtt1onary relattoll'>h1p between cave taxa and members of the Gcmmat1monadetes aOO Candidate D1\.JS1011 OPlO The deudrog:mm was constructed from an alignment of 1000 nucl-;otKle posllioir;: Distances were calcuJated m DNADIST and trees were mfem:d by the netgbbour-101nmg method 1Ttermoprota1s /enax U.iLS used as the ontgroup species The scale hu rnd1cates 10% sequence divergence

125 Chapter 3: Results and Discussion Nitrospira

One phylotype from saturated Loons sediment, SL20060, was similar to Nitrospira sp.

All known members of Nitrospira are obligate nitrite-oxidising chemolithoautotrophs (Ehrlich et al. 1995). Sequence information for clone SL20060 was only 300 bp in length and not included in further phylogenetic analysis.

126 Chapter 3: Results and Discussion I Euryarchaeota

Thermophilic Desulfurococcus amylolytzcus AF25033 l Crenarchaeota

Unidentt fled er en arch aeota isolate OCl 1 X 99561

Unculntred archaeonW SB-11ABO55993

Wetland soil Unculnired archaeon OS- 4 AB056029 clones

Uncultured archaeo n AM -17 A BO 56020

Lechuguilla Cave group South African Gold Mine group

~----Uncultured archaeon SAGMA- A AB050205 Mesophilic Manne Group I

Uncultured archaeon SAGMA- 2 AB050233 Mesophilic Terrestnal Group I Uncultured archaeo nE Al F4 0 I

Figure 3.18: Phylogenetic dendrogram illustratmg the evolu!Ionary relauonslnp between cave clone SL20017 and members of the Crenarchaeota. The dendrogram was constructed from an alignment of 1OOO nucleotide positions. Distances were calculated m DNADIST and trees were mferred by the ne1ghbour-Jommg method. Methanobactenum fomuc1cum was used as the outgroup species. The scale bar indicates 10% sequence divergence.

Archaea

One archaeal clone sequence, SL20017, was detected from saturated sediment from

Loons Cave (Figure 3.18). SL20017 is a deeply branching novel lineage only distantly related to cultured Crenarchaeota, Desulfurococcus amylolyticus; (75% sequence similarity) and most closely related to uncultured archaeon clones, WSB-11 (91.8%), OS-4 and AM-17 from wetland soils

(published only in datebase; Utsumi et al. 2001). All cultured Crenarchaeota including,

Desulfurococcus amylolyticus, are extreme thermophiles found in high temperature environments

(>80 °C). In recent years, nonthermophilic Crenarchaeota sequences have been detected from low to moderate temperature (1.5 to 32 °C) terrestrial and aquatic environments (-1.5 to 32 °C).

Mesophilic Crenarchaeota sequences were first reported from the Pacific Ocean (Fuhrman et al.

127 Chapter 3: Results and Discussion 1992). To date there have been no reports of successful laboratory pure cultivation of mesophilic

Crenarchaeota and nothing is known of their physiology and biochemistry (Northup et al. 2003).

There are four main clusters of mesophilic Crenarchaeota (Marine Group I, Freshwater cluster,

Terrestrial Group and FFSB cluster) that form a distinct lineage from thermophilic Crenarchaota.

Recently it has been proposed that a fifth cluster within the mesophilic Crenarchaeota clade is distinct and unique to the subsurface environment (three clones from South African gold mine waters; SAGMA clones) (Takai et al. 2001).

Clone SL20017 and the wetland soil clones do not branch with mesophilic sequences but rather form a separate clade more closely related to thermophilic Crenarchaeota. Almost all of the cultivated thermophilic Crenarchaeota are obligate anaerobes with sulfur-dependent metabolisms

(Buckley et al. 1998). The great phylogenetic distance of SL20017 to any cultured members of the

Crenarchaeota makes it difficult to infer any metabolic properties of this clone.

There have been few reports of archaeal cave communities at circumneutral pH, though those reported are suprisingly abundant and diverse including representatives of mesophilic

Crenarchaeota most closely related to organisms from marine or soil habitats, and 'Group 2'

Euryarchaeota (Mattison et al. 1998; Northup et al. 2003; Chelius & Moore, 2004). Overall, reported

Crenarchaeota sequences detected in cave environments cluster with those found in waters from a South African gold mine (SAGMA clones) (Takai et al. 2001) even though they have been detected from very different microhabitats, ferromanganese corrosion residues in Lechuguilla

Cave, New Mexico (Northup et al. 2003) and saturated sediment from Wind Cave, South Dakota

(Chelius & Moore, 2004). SL20017 does not align closely with any SAGMA or cave archaeal sequences. To draw too many conclusions on archaeal diversity on the basis of one clone sequence is too presumptive. This study would benefit from further culture-independent investigations employing Archaea-specific primers, as used in the previously mentioned cave studies, to target the archaeal portions of the microbial community.

128 ChaEter 3: Results and Discussion

Table 3.3: Taxonomic affiliations of cave isolates as determined by 165 rRNA gene sequencing. Presence of an isolate in a microhabitat represented as(+)

Isolate Taxonomic Identiftcahon or Nearest Cultivated SEE SPE SEL SPL ME2 ME3 MXl* Neighbour for Putatively Novel Species (%16S rRNA gene seguence sirmlan!}'.) ACTINOBACTERIA Micrococcineae CAVI333 Agrococcus 1enensis (98.43%) + CAVI349 Agromyces ramosum (98.79%) + CAVI005 Arthrobacter chlorophenolzcus (97.5%) + + + + + + + CAVI318 Arthrobacter pascens (99 .72%) + + + CAVI315, 002 Brachybacterium paraconglomeratum (98.69%) + + CAVI125 Brevibactenum iodinum (98.65%) + CAVI207 Knoellia sznensis (98.40%) + + + + CAVI006 Knoellza subterranean (98.34%) + + + CAVI117 Kocuria rosea (98.53%) + Corynebacterineae CAVI002 Nocardiaflumznea group (99.68%) + + + + + + + CAVI203 Rhodococcus erythropolzs (99.42%) CAVI104 Rhodococcus globerulus (99.27%) + + CAVI321 Rhodococcus wratislavensis (98.51 %) + CAVI306 Tsukamurella pulmonis (98.00%) + Pseudonocardineae CAVI0035 Amycolatopsis sp. nov. (A. sulphurea 96.90%) + CAVI018 Saccharothrzx sp. nov. (S albidocapillata 96.89%) + CAVI0051 Saccharothrzx cryophilus (98.23%) + + + CAVI312 Gen. Nov. [Saccharothrix violacea(92.94%); Lentzea + fiavoverrucosispora (92.93%)] Micromonosporineae CAVI0009 Couchioplanes caeruleus (96.65%); Actinoplanes + brasiliensis (96.95%) CAVI0023 Micromonospora sp. nov. (M purpureochromgenes + 96.82%) Streptomycineae CAVI004 Streptomyces aureus (99.50%) + CAVI308 Streptomyces beijzangensis (99.15%) + CAVI025, 116, Streptomyces caviscabies (99.08-99.82%) + + + + + 010, CAVI313, 019, Streptomyces caviscabies (97.76%) + + + 001 CAVI314 Streptomyces chattanoogensis (98.34%) + CAVI004 Streptomyces sp. nov. (S.clavulzgerus 97.37%) + CAVI328 Streptomyces microstreptospora (99 .10%) + CAVI231 Streptomyces sanglzeri (98.43%) + CAVI204, 003, Streptomyces subrutilus (98.02-99.86%) + + + + + + + 002, 006, 106 CAVI105 Streptomyces violceoruber (98.18%) CAVI005, 317 Kitasatospora mediocidica (97.57%) + + Firmicutes CAVI322 Bacillus sp. nov. (B cohnii 94.62%) + CAVI102 Bacillus sp nov. (B. pumilus 97.5%) + CAVI257 Bacillus cibus (99%) + CAVI007 Bacillus mycoides (97.5%) + CAVI008, 323, Bacillus simplex (98.25%) + + + + 275 CAVI320 Bacillus subtilus (98.75%) + CAVI309 Paenzbacillus sp. nov. (P. gramznis 97.47%) + CAVI319 Sporosarczna sp. nov. (S. macmurdoensis 97.34%) + CAVI106 Sporosarcina ureae (98.93%) + CFBs CAVI311 Flavobacterzum leeana (97.35%) + + CAVI120 Flavobacterzum psychrolimnae (98.20%) + + CAVI329 Gen. Nov. [Sphzngobacterzum multivorum (94.18%); + Pedobacter cryconztzs (95.15%)] CAVI339 Gen. Nov. [Sphzngobacterzum multivorum (95.90%), + Pedobacter cryconitis (96.49%)] CAVI317 Pedobacter cryconztzs (98.36%) + a-Proteobacteria

Continued on next page 29 Chapter 3: Results and Discussion

CAVI334 Ammobacter ni1gataens1s (99 .31 %) + CAVI210 Bosea thwox1dans (99.67%) + CAVI302 Brevundimonas alba (99.07%) + + CAVI326 Methylobacterzum sp. nov. (M.fuj1sawaense 95.45%) + + CAVI005 Sphmgomonas sp. nov. (S. aerolata 97.34%) + CAVI322 Sphmgomonas sp. nov. (S. melonzs 96.03%) + CAVI008 Paracoccus sp. nov. (P solvent1vorans 96.13%) + CAVI338 Porphyrobacter sp. nov. (P nuestonens1s 97.20%) + P.. Proteobacteria CAVI109 Acal1genes sp. nov. (A.faecalzs 96.05%) + + CAVIlll Acalzgenes sp. nov. (A. faecalzs 97.22%) + ~Proteobacteria CAVIllO Stenotrophomonas sp. nov. (S maltophila 97.21 %) + CAVI335 Xanthomonas sp. nov (X cameestris 95.12%) +

* Microhab1tats represented by samples: SEE (sediment, Entrance Cave), SPE (speleothems, Entrance Cave), SEL (sediment, Loons Cave), SPL (speleothems, Loons Cave), ME2 (calcite mat, Entrance Cave), ME3 (moommlk, Entrance Cave), and MXl (moomrulk, Exit Cave).

3.4 Isolation of Novel Cave Microbes

Cultures were isolated from sediment, speleothem and moorunilk samples from

Entrance-Exit Caves and Loons Cave to investigate culturable diversity (discussed in Section 3.2

and 3.5) and to determine the novelty of cave microbes. Caves are unique ecosystems exposed to

extreme environmental stresses. The limiting environmental characteristics of caves, little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate,

provide ecological niches for highly specialised and very diverse microbiota. Thus, this study

attempted to identify putatively novel cave microbiota. In accordance with the definition of a bacterial species, cave isolates with 2".: 97.5% 16S rRNA gene sequence similarity to validly

described microorganisms were considered to belong to the same species (Stackebrandt &

Goebel, 1994; Vandamme et al. 1996). Table 3.3 summarises the phylogenetic affiliations of representative isolates including putatively novel species and genera. A total of two putatively novel genera and 18 putatively novel species were identified.

Sediment and speleothem isolations were selective for actinomycetes, accordingly,

actinomycetes dominated the culture collection. However, three non-actinomycete isolates were identified including a putatively novel Paracoccus sp. isolated from a speleothem in Entrance

Cave. The majority of cave isolates from sediments and speleothems in both Entrance and Loons

Caves proved to be cosmopolitan members of the actinomycetes, particularly of the genera

Streptomyces, Arthrobacter and Nocardia. A number of novel actinomycete isolates were detected

130 Chapter 3: Results and Discussion from Entrance Cave sediments. One novel species, CA VI009 is phylogenetically most closely related to species from two genera, the Actinoplanes and Couchioplanes (Table 3.3). The boundaries of these genera is not clearly defined, Actinoplanes brasiliensis clusters with

Couchioplanes caeruleus rather than with other members of the Actinoplanes and isolate CAVI009 clusters within this clade (Figure 3.9b). Further characterisation of this novel cave isolate represents an opportunity to clarify the taxonomic positions of these species. Novel

Micromonospora sp. and Amycolatopsis sp. were also isolated from Entrance sediments and a novel Saccharothrix sp. was isolated from a speleothem in Entrance Cave.

Moonmilk isolations (which were non-selective) produced the most novelty, particularly from the Firmicutes, Proteobacteria, and CFBs. This result is not surprising given the uniqueness of the moonmilk habitat and the paucity of published studies of moonmilk microbes. Several isolates from all moonmilk samples belonged to the Gram-positive Firmicutes, particularly of the genus Bacillus (Table 3.3). There were four isolates deemed putatively novel species: two Bacillus sp. most closely related to B. cohnii and B. pumilus, a novel Paenibacillus sp. and a novel

Sporosarcina sp. most closely related to Sporosarcina macmurdoensis. The novel Sporosarcina sp. was closely related to an uncultured permafrost bacteria (Figure 3.14), indicating that this isolate is most likely a cold-adapted bacterium. Of the Proteobacteria, eight novel species were isolated

(Table 3.3). From the a-Proteobacteria a novel Methylobacterium sp., Porphyrobacter sp., and two

Sphingomonas sp., were detected. Two novel Alcaligenes sp. of the ~-Proteobacteria and a novel

Stenotrophomonas sp. and Xanthomonas sp. of the y-Proteobacteria were also isolated.

Demonstrating the novelty of the moonmilk cultures, members of two putatively novel genera were isolated. From sample ME3 two isolates, CAVI339 and CAVI329, most likely representing individual species (>2.5% sequence dissimilarity), clustered on a distinct branch within the

Sphingobacteriales of the CFBs (Figure 3.14). An uncultured bacterium detected in a karstic aquifer also branched within this clade. Isolates CAVI339 and 329 showed only 94-95% sequence similarity to Pedobacter sp. and Sphingobacterium sp., indicating that these two isolates from moonmilk may represent two species of a new genera within this lineage. Sample ME2, the calcite mat, produced an isolate, CAVI218 that clustered within the Pseudonocardineae but was

131 Chapter 3: Results and Discussion only distantly related to described members (Figure 3.9). CAVI218 showed the highest similarity

(-92%) to Saccharothrix violacea and Lentzea fiavoverrucosispora most likely representing a new

genus within this lineage.

The definition of a bacterial species used in this study (2 97.5% sequence similarity) is

considered by some to be too conservative, especially in the case of highly related species of

genera like Streptomyces. Also, 16S rRNA gene results are not sufficient alone to define a new

bacterial genus or species. Further morphological, biochemical and physiological testing, and

further genetic characterisation (% G+C, DNA:DNA hybridisation) is needed to validly describe

these putatively novel cave microbiota.

3.5 Differences in Microhabitat Community Structure

Measures of diversity were determined followed normalisation of the clone libraries

using the rarefaction method. Indices indicating biodiversity coverage (C), diversity (Shannon­

Weaver index H'). dominance (Simpson index SI'), and eveness (J') are displayed in Table 3.4.

Biodiversity coverage (C) (Mullins et al. 1995) measures the portion of a clone library of infinite

size that would be sampled by the smaller clone library obtained. The coverage of biodiversity was quite high for all libraries, ranging from 67.6 to 81.3% and particularly high for calcite-based

samples (76.1-81.3%).

132 Chapter 3: Results and Discussion

Table 3.4: Biodiversity indices for cave sediment and moonmilk samples.

Sample c H' SI' J'

SEl 71.8% 1.527 0.022 0.953

SE2 70.5% 1.400 0.030 0.914

SLl 70.5% 1.501 0.025 0.944

SL2 67.6% 1.494 0.025 0.939

ME2 76.1% 1.325 0.058 0.889

ME3 77.4% 1.379 0.024 0.943

MXl 81.3% 1.467 0.044 0.993

Estimates of Diversity (H') were determined using the Shannon-Weaver (or Shannon­

Weiner) Index (Krebs, 1989). This index measures the average degree of uncertainty

(synonymous with diversity) of predicting the species (or phylotype) of a given individual picked at random from a community. Diversity measures were high for all samples illustrating the diverse nature of cave microbial communities. Diversity was higher for the dry sediments from Entrance and Loons Caves (1.527 and 1.501 respectively) than the wet sediments (1.400 and

1.494 respectively). Dry cave substrate typically yields a higher proportion of Actinobacteria than

does dripping water and wet sediment (Kolbel-Boelke et al. 1988; Laiz et al. 1999). Perhaps the discrepancy in diversity measures may reflect the absence of high numbers of Actinobacteria from wet sediment. Dominance values were fairly low for all samples (0.022 to 0.058) and consequently eveness values were high (0.889 to 0.993). Measures of dominance concentration were determined using the Simpson Index (SI') (Krebs, 1989). This index is based on the probability of drawing a pair of individuals of the same species from a sample. Equitability indices (J') were based on Shannon-Weaver index data. This index measures the eveness with which individuals are distributed among the species present in a sample. Though all dominance values were comparatively low, the highest values were seen for the calcite mat sample ME2

133 Chapter 3: Results and Discussion (0.058), reflecting the high percentage of clones distributed among a few phylotypes (eg. Bosea sp., Brevundimonas sp.; Table 3.2)

There are many ecological diversity measures, but their suitability for use with highly

diverse bacterial communities is unclear and seldom considered (Hill et al. 2003). Inherent limitations of molecular techniques, including extraction efficiency and PCR bias, mean that measures of abundance, diversity and richness may not be strictly accurate reflections of the

actual community structure. Species abundance models are useful, irrespective of coverage, because they address the whole distribution of a sample, aiding comparison by revealing overall trends as well as specific changes in particular abundance classes. Bengtsson (1998) cautions that it is na'ive to contemplate that one single number - species richness, a diversity, the number of functional groups, or connection - can capture the complex relationships between many species

and the functions performed by these interactions in soil. The indices may reveal more if applied

to smaller, more homogenous habitats where a reasonable level of coverage may be obtained.

However, any estimates of microbial diversity must acknowledge the inability of microbiologists to satisfactorily define a bacterial species.

A graph of the distribution or abundance of major cloned phylogenetic groups (Figure

3.19) further highlights the similarities and differences in community composition over the range of samples, microhabitats, examined. Examination of the rRNA genes recovered from soil microbial communities at diverse sites reveal that eight bacterial groups are present in the majority of soil microbial communities: the a-, f3-, and y-Proteobacteria, the Actinobacteria, the

CFBS, the Acidobacteria, the Planctomycetales and the Verrucomicrobia (Buckley & Schmidt, 2001).

Cave sediment samples in this study were characterised by the presence of these typical terrestrial bacteria though in different abundances to other terrestrial environments (Figure

3.19). All clonal samples are dominated by the super-phyla Proteobacteria (39.4-77% of total diversity), however there are marked differences in the distribution of diversity between the a-, f3-, y- and o-divisions. Sample SEl showed striking differences to samples SE2, SLl and SL2. The latter were dominated by the f3- (24.6 %, 32.4% and 34.8%) and y- (26.2%, 25% and 14.1%)

Proteobacteria whereas SEl was dominated by the y-Proteobacteria (29.6%) and the f3-

134 Chapter 3: Results and Discussion Proteobacteria were not detected. The second striking difference is the abundance of

Actinobacteria (26.8%) and Planctomycetales (22.5%) in sample SEl compared to their comparatively lower presence in other sediments. Sample SEl is a dry sediment sample from

Entrance Cave and whereas samples SE2 and SL2 are saturated sediment samples which is the most probable explanation for the differences in community structure. Sample site SLl, though above the water line and chosen for its dry nature, is still likely to be more hydrated than sample

SEl due to the 'wet' nature of Loons Cave. The saturated sediment from Loons is more diverse than all other samples with members of 12 major phylogenetic groups being detected in comparison to 8-9 in other cave samples (Figure 3.19). A number of minor components detected in sample SL2 were not detected in other samples (eg. Gemmatimonadetes, Candidate Division

OPlO, Nitrospira and Crenarchaeota). These tended to be 'rare' phylotypes consisting of one clone and representing putatively novel lineages within divisions with few or no cultivated representatives (Figure 3.17, Figure 3.18). Members of these divisions are often detected in saturated, anoxic environments (eg. wetland soils, deep sea sediments, activated sludge).

Sample ME2, from the ceiling rock of Entrance Cave, displayed a very different community composition to all other samples, in particular, very different to calcite moonmilk samples ME3 and MXl. The a-Proteobacteria and Actinobacteria completely dominated the diversity sampled from ME2 (47.1 % and 37.1 % respectively). Within these major phyla, clones are distributed between few phylotypes within the genera Brevundimonas and Bosea and

Saccharothrix (Table 3.2) illustrating the simplicity of this mat-like microbial community.

135 Chapter 3: Results and Discussion

Figure 3.19: Comparison of community structure between cave microhabitats.

100%

90% • Archaea • Nitrospira 80% • Candidate Division OP10 Gemmatamonadetes 70% a C Verrucomicrobia 60% C Firmicutes • Chloroflexi 50% a Planctomycetales 40% • Acidobacteria CCFB 30% • Actinobacteria a Gamma Proteobacteria 20% C Delta Proteobacteria 10% • Beta Proteobacteria CAlpha Proteobacteria 0% SE1 SE2 SL1 SL2 ME2 ME3 MX1

The abundances of various cloned prokaryote groups in sediment and moonmilk samples from Entrance-Exit Cave system and Loons Cave calculated as percent of total clones. SEl - dry sediment, Entrance Cave. SE2 - saturated sediment, Entrance Cave. SLl - dry sediment, Loons Cave. SL2 - saturated sediment, Loons Cave. ME2 - calcite mat, Entrance Cave. ME3 - moonmilk, Entrance Cave. MXl - moonmilk, Exit Cave.

Calcite moonmilk samples ME3 and MXl show remarkable similarities in community structure (Figure 3.19) though being derived from geographically separated sites in Entrance and Exit caves. Also, their habit is quite different, ME3 was sampled from a moonmilk deposit beneath sediment, whereas MXl was sampled from a moonmilk-coated stalactite. Both samples are dominated by the ~-Proteobacteria (33.9% and 26.7% respectively), particularly the

Oxalobacteriaceae (Figure 3.6) whereas no ~-Proteobacteria taxa were detected in sample ME2.

Surprisingly, the second most abundant group in ME3 and MXl were the CFB (22.6% and 28% ), reinforced by the isolation of a number of Flavobacteriaceae moonmilk samples (Figure 3.15) however the CFB group were present in very low numbers in the mat material ME2 (1 .4 %). The fundamental difference between cave sediments and moonmilk samples was that the CFBs were

136 Chapter 3: Results and Discussion a comparatively minor component of cave sediment microbial diversity (2.8-9.8%). The

Actinobacteria, historically thought to be a major component of moonmilk based on culture­ dependent and microscopical studies, were the third most abundant clonal group (17.7% and

13.3%) though dominating the isolations (Table.3.3). The single similarity between sample ME2 and ME3 and MXl is the dominance of phylotypes affiliated with the Pseudonocardineae and

Micrococcineae (Table 3.2). The y-Proteobacteria were present in moderate numbers in sample

ME3 and MXl (12.9% and 6.7%) however not detected in sample ME2, again highlighting the differences between ME2 and moonmilk samples.

Pairwise comparisons of clone libraries were carried out using the Similarity Coefficient

(S) (Odum, 1971) which illustrates that the biodiversity of all sampl~s overlap, ie. share phylotypes, to some extent (Table 3.5). Moonmilk samples ME3 and MXl showed the highest similarity of comparisons (0.493). These samples shared 17 phylotypes reflecting the similarity of moonmilk microbial community composition in both Entrance and Exit Cave. The lowest similarity values were seen between sample ME2 and the Loons Cave sediment (0.086), sharing only three phylotypes. Sample ME2 was also distinct from Entrance Cave sediment (0.141 and

0.154) and moonmilk samples (0.133 and 0.141), highlighting the unique nature of the microbial community inhabiting the calcite mat. Sediment samples from Entrance Cave, SEl and SE2 shared a high number of phylotypes (15) as reflected by the high comparison value (0.405).

Similarly Loons Cave sediments shared 15 phylotypes reflected in the high similarity value

(0.385). Similarity values between Entrance and Loons Cave sediments are much lower indicating that the microbial communities are more similar within the individual cave systems them they are between similar habitats.

137 Chapter 3: Results and Discussion

Table 3.5: Pairwise comparisons of cave sediment* and moonmilk* clone library phylotype composition.

SE1 SE2 SLl SL2 ME2 ME3 MX1 SE1 1.000 SE2 0.405 1.000 SLl 0.177 0.356 1.000 SL2 0.152 0.247 0.385 1.000 ME2 0.141 0.154 0.086 0.086 1.000 ME3 0.203 0.159 0.118 0.147 0.133 1.000 MX1 0.100 0.162 0.228 0.228 0.141 0.493 1.000

* SEl - dry sedunent, Entrance Cave. SE2- saturated sediment, Entrance Cave. 511 - dry sediment, Loons Cave. SL2 - saturated sediment, Loons Cave. ME2- calcite mat, Entrance Cave. ME3 - moonmilk, Entrance Cave. MXl - moonmilk, Exit Cave.

3.6 Culturable vs. Non-culturable Diversity

It is widely recognised that culture-dependent techniques are limited and it has been estimated that less than 1% of the microorganisms in an environment are readily cultivated in the laboratory using standard techniques (Amann et al. 1995). The use of culture-independent molecular techniques to identify unculturable microbial species has vastly expanded our knowledge and understanding of microbial diversity (Pace, 1997). However, phylogenetic information does not necessarily impart information on the functional potential or in situ activities of microorganisms demonstrating an apparent need for characterising novel genera in pure culture to understand their functional role in the ecosystem. Actinobacteria are the most abundantly isolated group of bacteria from almost all published cave culture studies (except sulfur caves) of sediments, rock, speleofuems and rock art, seeming to demonstrate their dominance in these habitats (Groth et al. l999a; Groth & Saiz-Jimenez, 1999, Chelius & Moore,

2004). However, the apparent dominance of Actinobacteria in these habitats appears to be an artefact of culture-dependent studies. It has repeatedly been demonstrated in culture- independent studies of the same sites, that Actinobacteria do not dominate, and are a minor, to moderate at best, component of the community (Groth et al. l999a; Groth et al. 2001;

Schabereiter-Gurtner et al. 2002a, b; Chelius & Moore, 2004). The discrepancies between culture- dependent and culture-independent studies in relation to actinomycete abundance are not

138 Chapter 3: Results and Discussion restricted to cave environments. Li et al. (1999) isolated 75 different actinomycetes from marine samples; however very few actinomycete sequences were cloned from these same samples in a later study (Colquhoun et al. 2000; 1998a,b). Thus, this study endeavoured to determine how consistent concurrent culture-dependent versus culture-independent results were within the

Actinomycetales, rather than at the whole community level, by selectively isolating these bacteria from sediments and speleothems from Entrance and Loons Caves.

Actinomycetes were cultivated from all sediments using the selective procedures detailed in Section 2. Primary plates were dominated by actinomycete-like colonies with diverse morphologies and 165 rRNA gene sequencing revealed that isolates were distributed over eight genera. Approximately 60% of isolates were of the genus Streptomyces belonging to only a few species (Figure 3.11). Nocardia sp. were the next most abundantly isolated. Other genera isolated but in relatively low numbers were, Arthrobacter, Knoellia, Micromonospora,

Couchioplanes/Actinoplanes, Amycolatopsis and Saccharothrix. In contrast, though phylotypes affiliated with all these genera were detected in sediments, the relative abundances were not the same (Table 3.2). Most obviously, phylotypes affiliated with Streptomyces did not account for

60% of actinomycete diversity. Other taxa, not detected by cultivation, were detected in the clone analysis, eg. Frankia sp., Blastococcus sp., and Rhodococcus. These results demonstrate that culture studies of cave sediments, which we know are not representative at the whole community level, do not give a true representation of the actinomycete diversity either. Thus the observed cultured actinomycete diversity is more likely due to the ease with which members of the Actinomycetales can be cultured, eg. Streptomyces generally being the most easily cultivated actinomycete and thus represented by 60% of isolation results in this study.

There are very few published culture studies of moonmilk samples and no culture­ independent reports. Thus non-selective procedures were used to investigate culturable vs. non­ culturable diversity at the whole community level of moonmilk samples. Mostly novel isolates of the a-, 13- and y-Proteobacteria and CFBs and previously described Firmicutes dominated the culture collection of samples ME3 and MXl, rather than actinomycetes. Information on moonmilk microbial communities is scarce though isolations from moonmilk from several caves

139 Chapter 3: Results and Discussion in South Wales produced eight species of heterotrophic bacteria belonging to the genera Bacillus,

Micrococcus, Bacterium, and Streptomyces and moonrnilk consisting of a silicate gel in Nikitsky

Catacomb, Moscow, produced Flavobacterium sp., Alcaligenes sp. and Arthrobacter sp. (Williams,

1959; Semikolennykh, 1997). Speleothem dripping waters are probably a similar microhabitat to the very hydrated nature of moonmilk. Laiz et al. (1999) investigated the microbial diversity of dripping waters of Altamira Cave, Spain. Water communities were not dominated by actinomycetes but contained low proportions of Gram-positive bacteria, and were mainly composed of Gram-negative rods and cocci (Enterobacteriaceae and Vibrionaceae; genera

Aeromonas and Acinetobacter). Compounding this, in an earlier study of dripping waters in

Altamira Cave carried out by Somavilla et al. (1978) Bacillus and Pseudomonas appeared to be the most abundant genera, followed by Flavobacterium and Erwinia. The absence of culturable actinomycetes in dripping waters agrees with the observations of Kolbel-Boelke et al. (1988).

They found very few actinomycetes in 60 water samples clearly demonstrating that dripping water communities are very different to those of cave rock though both are heterotrophic based systems. This trend was reflected in the clone library analysis; moonmilk libraries being dominated by ~-Proteobacteria and CFB clones, accounting for more than 50% of the diversity sampled in total (Table 3.2, Figure 3.19). The Actinobacteria were far less dominant, (13-17% of diversity). An anomaly in clone and DGGE analysis was the absence of sequences related to

Bacillus species, though these were cultivated in great numbers from all moonmilk samples. PCR bias against Gram-positive, low G+C bacteria (Firmicutes) and the work of Laiz et al. (2003) who found that Bacillus species were not easily separated in DGGE analyses because of co-migration of bands which may explain this anomaly.

In contrast to moonmilk samples ME3 and MXl, isolations from sample ME2 were dominated by actinomycetes. This is not surprising given the abundance of clones affiliated with the Actinobacteria (37.1 %) and the networks of hyphal organisms visualised with ESEM. Which further demonstrates the uniqueness of the microbial community in the calcite mat in Entrance

Cave. The one common trend between the culture-dependent and culture-independent studies of the calcite mat and moonmilk microbial communities is the striking consistency between

140 Chapter 3: Results and Discussion isolations, clone phylotypes and DGGE phylotypes of the Actinomycetales (as previously demonstrated for sediment samples) and the CFBs and a.-Proteobacteria. Saccharothrix cryophilus,

Arthrobacter chlorophenolicus, Brevundimonas alba, Bosea thiooxidans and Sphingomonas sp. were detected in the isolations, clone libraries and DGGE analysis of all three samples. Flavobacteria leeana-like microbes were detected in isolations, clone libraries and DGGE analysis of moonmilk samples ME3 and MXl (CFBs were only a minor component of sample ME2).

Most published studies of isolations in caves have been performed using standard isolation procedures and incubation at 28 °C (Groth et al. 1999a, 2001; Laiz et al. 1999, 2000).

However, the constant low temperatures throughout the year in most studied caves suggests the possibility of an indigenous pychrophilic microflora, adapted to low temperatures, that could be overlooked using standard microbiological procedures with incubation at higher temperatures.

Laiz et al. (2003) isolated bacteria from sediments of Tito Bustillo, Llonin and La Garma Caves at a variety of temperatures from 5 - 45° C and investigated their temperature ranges for growth.

They found that isolated bacteria were psychrotrophs (Morita, 1975) or psychrotolerants, as most of them could grow at 5° C. No isolates had an optimum growth temperature below 20° C and therefore could not be considered true psychrophiles. The main difference in diversity of isolated bacteria with the use of different isolation temperatures concerned the recoverability of actinomycetes (Laiz et al. 2003). For example, at 13° Conly six actinomycete strains were isolated from Tito Bustillo though the diversity of non- actinomycete sp. increased in comparison to isolations at higher temperatures. At 28° C the number of actinomycete strains isolated was tripled, indicating that the isolation of actinomycetes diversity is temperature-dependent. Thus

Laiz et al. suggests the need to use low temperatures to detect maximum diversity of culturable bacteria other than actinomycetes and higher temperatures to detect maximum diversity of actinomycetes. Isolations in this study were carried out at 25 °C to detect maximum diversity of actinomycetes and at 10 °C to mimic the cave environment. Similarly to the results of Laiz et al.

(2003), most actinomycete diversity was detected in isolations from the calcite sample ME2 incubated at 25 °C whereas, at 10 °C only two colony morphologies were detected, one identified as Bosea thiooxidans. Conversely, there was no detectable difference between

141 Chapter 3: Results and Discussion incubation temperatures and colonies isolated for moonmilk samples ME3 and MXl;

Proteobacteria, CFBs and Firmicutes dominated all isolations reinforcing their dominance in moonmilk samples.

Through the course of isolation studies of moonmilk samples, a flaw was detected in earlier sediment and speleothem culture studies. Hyphal soil actinomycetes often show distinctive morphologies that can aid in identification to the generic level. These morphological characteristics include presence of aerial spores or mycelia that aids detection of "actinomycete­ like" colonies on primary plates. Culture studies of sediments and speleothems were aimed at sub-culturing only actinomycete-like colonies whereas culture studies of moonmilk samples were aimed at sub-culturing all different colonies. 165 rRNA gene sequencing revealed that many moonmilk colonies deemed "non-actinomycete-like" were actually actinomycetes (in particular members of the Micrococcineae and Corynebacterineae). Thus, a portion of the culturable actinomycete diversity may have been over-looked in isolations from cave sediments and speleothems.

The well recognised discrepancies between culturable and non-culturable diversity has limited our understanding of species diversity in natural bacterial communities. Plating leads to an overestimation of the number of spore-forming bacteria with respect to quiescent vegetative forms; the later are less easily cultured but readily detected by culture-independent techniques

(Laiz et al., 2003). Members of the Actinobacteria have established a wide ecological distribution and survive long periods of nutrient deprivation by producing endospores, which allows their ready cultivation in favourable conditions and the presence of available nutrients. However, the majority of oligotrophic organisms don't employ such sophisticated techniques to survive extreme nutrient deprivation, but simply grow and reproduce continuously at an exceedingly slow rate (Koch, 1997). A problem with cultivation of oligotrophs therefore comes with the assumption that the rate-limiting step in bacterial growth is simply nutrient availability, and not the ability of the cell itself to grow. The sudden addition of excess nutrients through cultivation methods to organisms adapted to nutrient limitation, may result in rapid cell death via osmotic swelling (Koch, 1997) thus many of these oligotrophic species cannot be easily cultivated using

142 Chapter 3: Results and Discussion standard techniques. Thus microbiologists need to work on developing culturing methods that better mimic the in situ chemical and physical parameters faced by microbes in the real world.

This limitation has been partially overcome by the advances of culture-independent techniques which have revealed surprisingly high levels of novel biodiversity. Some of these groups previously undetected by cultivation have emerged as numerically abundant and seemingly important ecological groups (eg. Acidobacteria). The limited number or absence of cultivated members of these groups restricts our understanding of their role in the environment. Parallel

studies of laboratory cultures strongly complement molecular ecological investigations. Recent

advances by Sait et al. (2002) and Joseph et al. (2003) have shown that many of the previously

uncultured lineages can be isolated using relatively simple media (Eg. the Ellin isolates of the

Acidobacteria, Verrucomicrobia, Gemmatimonadetes and novel members of already well characterised phyla, Proteobacteria and Actinobacteria).

3.7 Metabolic/Ecological Comparisons

By phylogenetically aligning an organism to its next nearest cultivated relative, we may shed light on the metabolic and physiological processes that are occurring (Pace 1997). However such comparisons can only be made when there is a high degree of sequence similarity between the identified phylotypes and known cultivated species (Achenback & Coates, 2000). Caves are severely resource limited due to the absence of light that precludes primary production of organic material by photosynthetic organisms (Northup & Lavoie, 2001). In cave ecosystems with little or no exogenous organic input, the rich variety of redox interfaces allows primary growth of chemolithotrophic (eg. ammonium-, nitrite-, sulfur-, manganese- or iron- oxidising) bacteria (Northup & Lavoie, 2001). Chemolithotrophs are physiologically united by their ability to utilise inorganic electron donors as energy sources. Most chemolithotrophs are also capable of autotrophic growth. The best studied chemolithotrophs are those capable of oxidising reduced sulfur and nitrogen compounds and the hydrogen-oxidising bacteria.

143 Chapter 3: Results and Discussion Chemolithotrophic growth on reduced sulfur compounds is a property of a diverse group of bacteria many of which were identified in clone analysis of both sediment and moonmilk samples in this study, particularly members of the Chromatiales that dominated the dry sediment from Entrance Cave. Sulfur-oxidisers mostly oxidise reduced sulfer compounds like sulfide and thiosulfate. The calcite mat-like material from Entrance Cave was dominated by a phylotype closely related to Bosea thiooxidans, thiosulfate-oxidiser, which was also isolated from this site. Sulfur-oxidising bacteria play a role in the dissolution of limestone in caves with hydrogen-rich waters, contributing to cave enlargement. The extent to which bacteria contribute to the corrosion of limestone and to the enlargement of existing caves remains uncertain. The sulfide needed by sulfur-oxidisers can be derived from sulfate- or sulfur-reducing bacteria associated with sulfur-oxidising bacteria. The o-Proteobacteria consists of sulfate and sulfur­ reducing bacteria. These microbes are obligately anaerobic and morphologically diverse. They are widespread in terrestrial and aquatic environments that become anoxic as a result of microbial decomposition processes, for example, Desulfovibrio species are common in waterlogged soils like Loons sediments, containing abundant organic material and sufficient levels of sulfate. The activity of sulfate-reducing bacteria leads to the production of large amounts of H2S. Some sulfate-reducers also display the ability to grow chemolithotrophically using ferrous iron at acid pH, eg. Acidithiobacillus ferrooxidans, sequences related to this species were detected in Entrance sediment.

A number of the Proteobacteria are nitrifying bacteria able to grow on reduced inorganic nitrogen compounds. No chemolithotroph is known to carry out the complete oxidation of ammonia to nitrate thus nitrification of ammonia in nature results from the sequential action of two separate groups of organisms: the ammonia-oxidising bacteria, nitrosifyers (eg. Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp.) and the nitrite-oxidising bacteria nitrifyers (eg. Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp.). Most nitrofyers are obligate chemolithotrophs and able to grow when provided with C02 as the sole carbon source. Nitrifyers are wide spread in soil and water though usually more abundant in neutral or alkaline habitats as acidity results in inhibition of nitrification. High identity values

144 Chapter 3: Results and Discussion with cultivated members of these groups, particularly members of the Pseudomonads and

Xanthomondales, may indicate that bacteria detected in all cave sediments and moonmilk analysed in this study may play a role in the nitrogen cycle. Clone analysis also revealed affiliations with aerobic nitrogen-fixing bacteria in cave sediments, including plant-associated genera (eg. Rhizobium, Bradyrhizobzum, Frankia) and free-living genera (eg. Derxia and

Beijerinckia). Interestingly, plant-associated nitrogen-fixers of the Oxalobacteriaceae and

Burkholderiaceae were particularly dominant in Loons sediments and moonmilk speleothems.

The waterway in Loons Cave is thought to be primarily fed by seepage waters through the ceiling rock. Filtration waters are also involved with the development of speleothems. The significant presence of plant-associated nitrogen-fixers in these samples may be a result of bacteria filtering with seepage waters into the cave systems from the surface soils.

Chemolithotrophic hydrogen-oxidising bacteria, including representatives in the genera

Pseudomonas, Paracoccous and Acaligenes, Hydrogenophaga, Acidovorax and Arthrobacter, are capable of growing with H 2 as the sole electron donor and 0 2 as the electron acceptor. Most hydrogen-oxidising bacteria are facultative and can also grow as chemoorganotrophs with organic compounds as energy sources. This represents the major distinction between hydrogen­ oxidisers and the nitrifyers and sulfur bacteria as most representatives of these groups are obligately chemolithotrophic and growth does not occur in the absence of the inorganic energy source. By contrast, the hydrogen chemolithotrophs can switch between chemolithotrophy and chemoorganotrophy and presumably do so in nature as nutritional conditions warrant. Several sequences obtained from both sediment and moorunilk were related to hydrogen-oxidising bacteria, particularly of the Acidovorax group and novel Paracoccus and Acaligenes species were isolated from a speleothem and moorunilk, respectively.

Early researchers proposed that the role of microbes in caves is to serve as a food source for higher trophic levels (Dickson, 1979). However it was typically believed that microbes could not provide adequate energy to support a large and diverse ecosystem. The work of Sarbu et al.

(1996) in Movile Cave, Romania, and by Vlasceanu et al. (2000) in Frasassi Caves, Italy, suggest that chemoautotrophic, sulfur-based microbial communities can generate enough energy as

·145 Chapter 3: Results and Discussion primary producers to sustain complex cave ecosystems. Thus it is proposed that the chemolithotrophic and oligotrophic bacteria identified in this study may support the abundant heterotrophic microbial life detected in all samples. However, heterotrophic cave microbial communities usually rely on allochthonous input of organic matter transported from the surface

(Groth et al. 1999a). Animals and visitors can provide large amounts of organic input facilitating heterotrophic life. Organic input may also be dissolved in the seepage/ dripping waters or as particulate organic matter carried in by active or periodic flooding of a subterranean streamway

(Schabereiter et al. 2002). Previous research has suggested that cave waters contain dissolved organic matter from the soil, primarily phenolic compounds and lignin (Saiz-Jimenez &

Hermosin, 1999). These compounds can be utilised as carbon sources by many of the species related to those identified in this study. High sulphate and nitrate concentrations have been found in dripping waters in Tito Bustillo and other Spanish and Italian caves (Hoyos et al. 1999) which, in addition to the concentrations of iron, manganese and other elements found in cave rocks, probably supports heterotrophic bacteria including members of the Actinobacteria, that were dominant members of the dry sediment and calcite mat microbial communities in Entrance

Cave, and members of the Flavobacteriaceae that were dominate community components of moonmilk samples.

In oligotrophic environments there are no obvious sources of exogenous energy sources

(eg. surface organics, sulfide or nitrite). A common theme was observed in cultivated relatives of identified phylotypes in this study: the fixation of atmospheric gases or the use of aromatic carbon compounds. Within the a-Proteobacteria phylotypes related to species able to fix atmospheric gases (eg. Sphingomonadales, Brevundimonas sp., Hyphomicrobium sp. and

Methylobacterium sp.), were particularly abundant in the calcite mat from Entrance Cave and were also detected in Loons sediments and moonmilk samples. Several sequences showing high sequence similarity with oligotrophic bacteria of the Sphingomonadales were detected in the calcite mat from Entrance Cave. Methylotrophs are chemoorganotrophs that utilise carbon

compounds more reduced than C02, are widespread in aquatic and terrestrial environments and include representatives of the genera Methylobacterium, Methylocella, and Hyphomicrobium.

146 Chapter 3: Results and Discussion Hutchens et al. (2004) used DNA-based stable isotope probing and functional gene analysis of groundwater and mat material from Movile Cave to identify methane-assimilating populations and results suggest that aerobic methanotrophs (Methylomonas, Methylococcus,

Methylocystis/Methylosinus strains) actively converted CH4 into complex organic compounds and thus helped sustain a diverse community of microbes in this closed ecosystem. Hyphomicrobium spp. are able to use atmospheric methyl-halides as their sole source of carbon and energy and are also able to oxidise manganese (McAnulla et al. 2001). A novel pink-pigmented

Methylobacterium sp. was isolated from moonmilk. Methylobacterium carry out Type I formaldehyde assimilation and this activity has previously been described in oligotrophic bacterial communities living on limestone masonry (Hanson & Hanson, 1996). The source of atmospheric gasses is clear, while the potential carbon sources may be the organic constituents of water filtering into the cave system. Northup et al. (2000) suggested that reduced metals such as magnesium and iron within the limestone matrix of Lechuguilla Cave provide sufficient source of electron donors for growth, which may further require the presence of atmospheric organic molecules as a carbon source. Similar mechanisms of lithotrophy have been suggested in other cave systems (Cunningham et al. 1995). However, moonmilk samples from Entrance and

Exit Caves are almost pure CaC03 (-98-100%) with no significant presence of reduced metal compounds available to act as electron donors. Similarly, the Leadville limestone bedrock of

Fairy Cave, Colorado, is almost pure CaC03 (97.5%). Barton et al. (2004) suggested that any metal ions present in Fairy Cave were likely deposited by the rich mineral waters that formed the cave system.

The physiological response of bacteria to temperature is critical for the regulation of biogeochemical processes. Moonmilk samples were found to harbour an abundant microflora of phylotypes and isolates closely related to described psychrotrophs which is not surprising given the near constant cold temperatures (7-10 °C) in Tasmanian caves. As discussed previously, Laiz et al. (2003) investigated temperature ranges of cave microbiota finding that though they were able to grow at 5 °C, growth optima were above 20 °C which indicates psychrotolerant growth, not true psychrophilic growth. There are conflicting views as to the effect of cold environmental

147 Chapter 3: Results and Discussion temperatures on the in situ chemolithotrophic metabolic rates of psychrotolerant bacteria. Zhang et al. (1999) found that as a physiological adaptation of natural microbial populations to the permanently cold deep Pacific marine sediments and Alaskan tundra permafrost, reduction of ferric iron utilising organic acids or hydrogen as electron donors, was fastest at 10 °C than at 25

°C, indicating that microbial iron reduction is likely widespread in cold environments.

Conversely, sulfate-reducing bacterium Desulfobacterium autotrophicum responded to low temperatures by reducing metabolic activities, which agrees with in situ activities measured in field studies and was suggested to reflect a common physiological principle of psychrotolerant bacteria (Rabus et al. 2002). Arnosti et al. (1998) found that rates of organic carbon mineralisation were always higher at temperatures above ambient environmental temperatures in Arctic and temperate sediments. However, as the mean environmental temperature dropped, the optimal temperature also dropped, suggesting that organic carbon turnover in the cold Arctic was not actually intrinsically slower than in temperate environments. One study of metabolic activities related to in situ temperatures for cave microbiota demonstrated that carbon utilisation was found to be more efficient at lower temperatures (13 °C) suggesting that these bacteria were adapted to live at lower temperatures than their optimal (Laiz et al. 2003). It is widely accepted that psychrotrophs are able to metabolise at lower than optimum temperatures and thus are able to continue growth in cold environments. Whether these organisms are metabolising at optimum rates at in situ temperatures remains unclear.

3.8 Comparison with other Cave Environments

Literature on cave microbial communities, their taxonomic diversity and distribution, is limited and restricted to a few caves worldwide. Culture-independent analyses have opened the way to study microbial diversity in environmental samples without prior cultivation more often than not revealing surprising diversity. Nevertheless, until recently, our knowledge of bacterial communities in caves has been largely due to culture-dependent studies. The beginning of this century has seen an influx of culture-independent diversity analyses of cave environments

148 Chapter 3: Results and Discussion enormously increasing our knowledge of cave microbial diversity. However, with the diverse range of types of caves (eg. sulfur caves, carbonate caves, aquatic caves, tourist/show caves, restricted access caves) and microhabitats (eg. acidic biofilms on walls, filamentous microbial mats in sulfur waters, aquatic microbial mantles, Palaeolithic rock art, cave walls, ferromanganese deposits, sediments) studied and the geographic separation of sites (Romania,

Italy, Australia, Mexico, Spain, North America) it can be difficult to draw comparisons or conclusions about cave microbial diversity (eg. Sarbu et al. 1996; Angert et al. 1998; Vlasceanu et al. 2000; Holmes et al. 2001; Summers-Engel et al. 2001; Schabereiter-Gurtner et al. 2002, 2004;

Northup et al. 2003; Chelius & Moore, 2004; Barton et al. 2004).

BLAST comparisons of sequences obtained in this study to the GENBANK database consistently yielded high similarity with several DGGE sequences from the Proteobacteria,

Actinobacteria, CFBs, Acidobacteria, Planctomycetales and Chloroflexi, detected in the analysis of rock art and surrounding cave surfaces in Spanish and Italian caves (Schabereiter-Gurtner et al.

2002, 2004). Universal primers 341£ and 907r were used in the DGGE studies by Schabereiter­

Gurtner & colleagues whereas primers 519f and 1492r were used in this study, thus there was only a 3-400 bp overlap between sequences obtained in either studies. Determining exact phylogenetic relationships between the rock art phylotypes and the clones from this study was often difficult and the DGGE sequences were removed from subsequent phylogenetic analysis.

Chelius & Moore (2004) stated that because few exhaustive studies of microbial community structure in caves have been conducted, habitat-specific trends are difficult to detect.

Although some clones they obtained from saturated cave sediments in Wind Cave resembled sequences from other caves, they found that no cave-specific bacterial community was evident.

Clones mostly resembled those from soil, freshwater, plant associated and polluted environments and most isolates were related to other cultivated members and sequences retrieved from soil and various polluted environments (Chelius & Moore, 2004). Similarly, aside from the cave rock art DGGE sequences, few cave sequences resulted from BLAST searches of the clones from this study. However, rather than being a function of the paucity of studies, it's hypothesised that perhaps these trends are difficult to elucidate due to the overwhelming

149 Chapter 3: Results and Discussion quantities of data available. With the explosion of culture-independent studies in the past 15 years, to date, more than 62 OOO sequences are available from public databases. Thirty seven division-level lineages have been detected (Hugenholtz et al. 1998), almost a third of which are not represented by cultured microorganisms. This provides a high resolution framework for the assignation of novel sequences obtained in 16S rRNA gene libraries constructed from environmental diversity surveys but perhaps disguises the phylogenetic groupings consistently found in caves. Results from this study indicate that there are some general and more specific trends apparent in cave samples.

There is much evidence for rich and diverse chemoautotrophic and heterotrophic microbial communities in caves. Several studies of chemolithotrophic cave microbial communities that do not depend directly upon energy and organic carbon from photosynthesis, have been reported and its been demonstrated that these bacteria play an important role in some cave ecosystems, acting as primary producers and supporting growth of heterotrophic microbes

(eg. Sarbu et al. 1996). High sulfate and nitrate concentrations have been found in dripping waters in Spanish and Italian caves (Hoyos et al. 1999; Van Grieken et al., 1999; Holmes et al.

2001) which, in addition to the concentrations of iron, manganese and other elements found in the cave fully support the finding of bacteria involved in the nitrogen, sulphur, iron and manganese cycles. Members of the Proteobacteria dominate all culture-independent analyses of - cave environments, including this study. Sulfur- and sulfide- oxidisers, iron- and manganese­ oxidisers, sulfate-reducers and nitrifiers and denitrifyers appear abundant in caves. Nullabor

Caves were found to have a high abundance of Nitrospira clones suggesting chemoautotrophic communities dependent on nitrite-oxidation (Holmes et al. 2001). A number of clones grouping with Nitrospira sp. were also found in ferromanganese deposits in Lechuguilla (Northup et al.

2003) and in Llonin, La Garma and Tito Bustillo Caves in association with Palaeolithic rock art

(Schabereiter et al. 2002, 2004). Schabereiter-Gurtner et al. (2002) found ammonia oxidisers such as Nitrosospira sp., and Nitrosococcus sp., in Tito Bustillo Cave. Clone library analysis by Chelius

& Moore (2004) illustrated that y-Proteobacteria and predominated water-saturated sediments in the dark zone of Wind Cave. A number of alpha phylotypes which grouped into two 'fixer'

150 Chapter 3: Results and Discussion clades are common in caves (eg. Chromatiales, Hyphomicrobium, Chelatobacter and Rhizobium).

These phylotypes are related to species able to fix atmospheric gases. Members of the

Comamonadaceae, particularly of the Acidovorax group have been detected in high numbers in ferromanganese residues of Lechuguilla and cave rock art (Northup et al. 2003; Schabereiter et al., 2004) and were particularly dominant in Loons Cave sediment and moonmilk.

The Acidobacteria have been detected as one of the most abundant groups of microbes in recent culture-independent analyses of a number of cave rock art sites and saturated sediment in

Wind Cave (Schabereiter-Gurtner et al. 2002, 2004; Chelius & Moore, 2004). Oones affiliated with the Acidobacteria were detected in most samples in this study (except for SEl) but in low numbers (1.5-3.2%). Both Wind Cave and the cave rock art sites are 'show' caves open to the public. With few cultured representatives of this group, little is known about ecology of

Acidobacteria. Perhaps the increased colonisation of Acidobacteria in show caves compared to more restricted access caves is due to the anthropogenic impacts and increased nutrient load connected with visitors to the caves. Minor representations of the Planctomycetales, the Chloroflexi

(green non-sulfur), particularly of the Dehalococcoides lineage, the Verrucomicrobia and the

Gemmatimonadetes (previously Candidate division BD), have been detected in a variety of cave environments (aquatic formations in Nullabor caves, Holmes et al. 2001; rock art and cave walls in Altamira and Tito Bustillo Caves, Schabereiter et al. 2002, 2004; saturated sediments in Wind

Cave, Chelius & Moore, 2004) mostly displaying low similarities to known, cultivated relatives of these groups suggesting new lineages. One point of interest is the high numbers (previously unreported for cave sediments) of the Planctomycetales (22.5%) in sediment sample SEl from

Entrance Cave. The Planctomycetales were considered to be of limited environmental importance, but molecular microbial ecology has demonstrated that these bacteria are ubiquitous, metabolically diverse and constitute a representative part of the natural bacteria population in diverse environments (Hugenholtz et al. 1998).

A significant result of this study is the abundance of cloned members of the CFBs in moonmilk samples ME3 and MXl (Figure 3.19). As previously stated, the CFBs, particularly

Flavobacteriaceae, were the second most abundant phyla in sampled moonmilk diversity. DGGE

151 Chapter 3: Results and Discussion and isolation results also confirm the dominant presence of these microbes (Figure 3.14). CFBs

have rarely been identified in previous cave studies (cultured or uncultured), and when present

are as minor components showing low similarity identities with known Cytophagales members

(Schabereiter-Gurtner et al. 2002; Chelius & Moore, 2004; Barton et al. 2004). Previous studies

identifying CFBs in caves have been based on sediment samples. Similarly, sediments from

Entrance and Loons Caves also displayed low CFB abundances (2.8-9.8%}. Due to the absence of

any published culture-independent analysis of moonmilk, it is impossible to determine whether

the dominance of CFB phylotypes is a general trend. Although isolations from moonmilk

consisting of a silicate gel in Nikitsky Catacomb, Moscow, produced Flavobacterium sp.,

(Semikolennykh, 1997).

Culture-dependent studies have focused on caves with allochtonous input of organic

matter demonstrating that heterotrophic bacteria dominate these microbial communities (Groth

& Saiz-Jimenez, 1999). Actinomycetes are the most abundant bacteria to be isolated from

heterotrophic cave systems, and have demonstrated great taxonomic diversity. Interestingly, in

most papers the recognition of this biodiversity, that is the identification of cave isolates, has

been through chemotaxonomic analysis only to the genus level. This has resulted in a lack of 16S

rRNA gene sequences of cave isolates in public databases, making it difficult to compare and

contrast biodiversity at the species level between other studied cave systems. Nevertheless we

can still determine trends at the genus level.

The most abundant actinomycetes isolated from caves are the streptomycete,

nocardioform and coryneform actinomycetes (eg. Groth et al. l999a; Chelius & Moore, 2004).

Samples of sediments, active stalactites, wall concretions and rocks from the walls and ceilings

of various caves have been investigated and a high number of isolates obtained. Most abundant

were the genera, Streptomyces, Nocardia, Nocardioides, Brevibacterium! Rhodococcus, and members

of the family Micrococcaceae. Similarly, in this study, members of the genera Streptomyces,

Nocardia, Arthrobacter and Rhodococcus were repeatedly cultivated in high numbers from

sediment, speleothem and moonmilk samples from Entrance, Exit and Loons Caves (Table 3.3).

Also isolated in this study, though less frequently, were Agromyces sp., Agrococcus sp., Knoellia

152 Chapter 3: Results and Discussion sp., Brevibacterium sp., and Brachybacterium sp. (Table 3.3). Members of the Pseudonocardineae, particularly of the genera, Lentzea, Saccharothrix, and Amycolatopsis, were the most abundantly isolated and culture-independently detected group of Actinobacteria from walls and rock art in

La Garma and Llonin caves (Schabereiter-Gurtner et al. 2004) and also from calcite-based samples in this study.

Many microbes identified from deep caves are similar to surface forms and are probably transported into caves by water, air, sediment and animals (Groth et al. 1999a; Groth et al. 2001;

Saiz-Jimenez 2001; Schabereiter-Gurtner et al. 2002a, b; Chelius & Moore, 2004) as reflected by the high sequence identity of isolates and clones to already cultured and cosmopolitan representatives of this group. However, recent findings and the results of this study have revealed the presence of actinomycete species so far only detected in caves. Knoellia sinensis and

Knoellia subterranean, isolated from sediment in Reed Flute Cave in China (Groth et al. 2002) was isolated and clones detected in cave sediment from Entrance Cave and all moonmilk clone libraries. Saccharothrix violacea isolated from a gold mine cave in China (Lee et al. 2000) was detected in clone analysis and isolated from sediment and moonmilk samples from Entrance

Cave and DGGE analysis of moonmilk and has also been detected in ferromanganese deposits in Lechuguilla and cave rock art. Recent studies have emphasised the unique nature of the bacterial and archaeal assemblages found in geographically separated and distinct 'types' of caves (Holmes et al. 2001; Schabereiter-Gurtner et al. 2002, 2004; Northup et al. 2003; Barton et al.

2004; Chelius & Moore, 2004). For example, a large proportion of the Crenarchaeota sequences detected in Lechuguilla Cave ferromanganese deposits and saturated sediments from Wind

Cave closely resembled sequences from a South African gold mine (SAGMA clones which showed great phylogenetic diversity to uncultivated members) (Takai et al. 2001; Northup et al.

2003; Chelius & Moore, 2004). Given the distance between North America and South Africa, it is unlikely that the archaeal assemblages are as similar by chance or by recent colonisation. A more plausible explanation is that the archaeal clones isolated from these sites evolved within a common subsurface environment, a conjecture supported by the geologic history of the respective regions and recent work on SAGMA clones (Chelius & Moore, 2004). Thus, in the

153 Chapter 3: Results and Discussion same way members of the genera Knoellia, Lentzea and Saccharothrix may represent indigenous cave microbiota present in a wide variety of global subterranean environments. Although it is not clear whether these trends represent convergent evolution at different geographical sites or whether these taxa represent remnants of ancient forms that existed when the continents were joined as the global super-continent, Pangea which formed over 600 million years ago (Mya) and began to separate approximately 400 Mya.

154 Chapter 4: Concluding Remarks

Chapter 4: Concluding Remarks

Over the last 15 years it has definitely been established that large and diverse microbial populations are active to great depths in the terrestrial subsurface and below the sea floor. One of the most compelling questions is the mechanism of supply of nutrients to subsurface populations. Both heterotrophic organisms that consume deeply buried ancient carbon and chemolithotrophic organisms that harness geochemical energy of reactive rocks have been documented in the subsurface. However abundant organisms do not exist everywhere in the subsurface, making it clear that a better understanding of the ecology of the subsurface ecosystems is needed to predict the abundance and significance of the subsurface biosphere.

Caves are not uniform environments in terms of geological and geochemical characteristics, as they can vary from one to the other, eg. rock type, method of formation, length, depth, number of openings to the surface, presence or absence of active streamways, degree of impact by human visitation etc. Furthermore, on a smaller scale, various microhabitats, with vast differences in community structure can exist within caves. Despite many recent advances in the field, literature on cave microbial diversity is still scarce and very few general trends have been detected. Literature on microbial diversity in caves has indicated that actinomycetes dominate culture studies. The majority of the work on actinomycetes in hypogean environments has been conducted in Altamira, Tito Bustillo, La Garma, and Llonin caves, Spain, and Grotta dei Cervi, Italy all of which have spectacular galleries with Palaeolithic rock art paintings, and more recently, in Wind Cave, Dakota, all of which are open to the public

(Groth & Saiz-Jimenez, 1999; Groth et al. 1999a, 2001; Laiz et al. 1999, 2000; Chelius & Moore,

2004). Culture-independent analyses in all of these show caves indicate that actinomycetes are not the most dominant member of the microbial communities.

Based on the literature available, this study was initially aimed at detecting novel actinomycete diversity. However, as the study progressed the focus evolved as it became apparent that actinomycetes dominated only very specific habitats, the dry sediment and the calcite mat in Entrance Cave, and represented only a minor fraction of most other microhabitats

155 Chapter 4: Concluding Remarks studied. Entrance Cave dry sediments and inactive (dry) speleothems produced a higher number of actinomycete isolates compared to saturated sediments and wet formations from

Entrance and Loons Cave which was reinforced by the actinomycetes being the second most abundant group (26.8%) detected in clone analysis of the dry Entrance sediment and low abundances (4-16%) detected in saturated sediments from both Entrance and Loons caves. Many actinomycetes are obligate aerobes and prefer moderate levels of moisture rather than waterlogged soils (Williams et al. 1972) and it seems fairly widely accepted that dry cave substrate typically yields a higher proportion of actinomycetes than does dripping water and wet sediment (Kolbel-Boelke et al. 1988; Laiz et al. 1999). Actinomycetes dominated isolations from the calcite mat in Entrance, and were a major component of the clone analysis, which is not surprising given the results of ESEM studies showing extensive networks of hyphal organisms that compose the mat. Moonmilk samples in comparison were not dominated by actinomycetes.

As a result of this work, it is hypothesised that the dominance of actinomycetes in the cave literature is due to the caves that these culture studies have been conducted in being show caves open to the public, providing increased allochtonous input, together with the organics available in the paint layers of rock art, leading to a proliferation of heterotrophic organisms. Secondly, it may be a function of the ease, rather than dominance, with which some actinomycetes can be cultured in the laboratory compared to other indigenous cave bacteria.

The Entrance-Exit Cave System and Loons Cave offer contrasting opportunities with regard to the search for novel microbial biodiversity, as do sediment samples versus moonmilk.

Sediment phylotypes and isolates identified in this study closely resemble species associated with oligotrophic, chemolithotrophic and heterotrophic lifestyles indicating that these communities survive by utilising a combination of metabolic pathways. Bacteria involved in the nitrogen and sulfur cycles were important members of all sediments along with hydrogen­ oxidising bacteria. These oligotrophic and chemolithotrophic members of sediment communities probably provide energy for the heterotrophic members of the community. Pair-wise comparisons of sediment communities demonstrated that they were more similar to each other within individual cave systems, Entrance and Loons, rather than between microhabitat types

156 Chapter 4: Concluding Remarks (dry vs. wet sediment). Indicating that the type of cave system does have an effect on the observed biodiversity, probably a reflection of differing factors such as nutrient supply. For example, in Entrance Cave there is the active inflow stream, Mystery Creek, which would bring exogenous inputs of nutrients into the system whereas Loons is fed primarily by ultra-filtered seep waters. The wet sediment from Entrance Cave did show a higher degree of similarity in community composition to Loons Cave samples than the dry sediment from Entrance Cave indicating that the water content of the sediment also has an affect on the distribution. Saturated sediments were dominated by oligotrophs able to fix atmospheric gases and methanotrophs and had a high proportion of rare phylotypes most likely representing new lineages related to microbes detected in anaerobic, anoxic environments, but low abundances of heterotrophic microbes.

Results also demonstrated a marked difference between sediment communities and calcite communities. One of the more significant findings in this study was the work with moonmilk. Results of ESEM and XRD analysis demonstrated that samples ME3 and MXl are true calcite moonmilk (mondmilch). Phylogenetic analyses and isolation results demonstrated the unique composition of the microbial communities associated with moonmilk deposits. These were predominantly composed of nitrogen-fixing ~-Proteobacteria and psychrotrophic heterotrophic CFBs and to a lesser extent, heterotrophic Actmobacteria. This study also revealed the dominant presence of cold-adapted (psychrotrophic) aerobic heterotrophic CFBs in moonmilk samples, indicating that this bacterial community survives utilising nitrite and organic material probably dissolved in the cave dripping waters. Phylogenetic analyses and biodiversity indices reveal the striking similarities between moonmilk samples from both

Entrance and Exit Caves and the uniqueness of the calcite mat in Entrance Cave. Despite XRD and ESEM analysis showing similar calcite composition and crystal morphology, phylogenetic results indicated that sample ME2 represented a very different microhabitat to moonmilk samples. The mat-like material of site ME2 was dominated by oligotrophic a-Proteobacteria and

Actinobacteria composing 84.2% of the total diversity. Though mostly a heterotrophic, community, members of the genus Saccharothrzx, present in high numbers in the calcite mat, are

157 Chapter 4: Concluding Remarks chemoorganotrophic. Metabolic analyses suggested that the community subsists using a complex metabolic network with input from trace organics within the environment or fixation of atmospheric gases using lithotrophic metabolism.

ESEM investigations of the ceiling rock and moon milk illustrated networks of hyphal bacteria involved with CaC03 crystallisation. The one significant similarity between the moonmilk samples and mat material was the dominance of Saccharothrix sp. in the clone library,

DGGE analysis and isolation results for all samples indicating that members of this genus are a dominant member of cave calcite microhabitats. Caii.averas et al. (1999) isolated a Saccharothrix sp. from calcite which grew very slowly and formed a deep black soluble pigment on oatmeal agar and developed a white aerial mycelium which turned grey with age. This morphology is similar to a culture isolated from samples ME2, ME3 and MXl and identified as Saccharothrix cryophilis. A clone phylotype and DGGE band also present in all samples were most closely related to this organism also perhaps indicating that these organisms are involved in calcite precipitation.

This study represents the first reported culture-independent analysis of moonmilk microbial communities globally and of cave sediment communities in the Southern Hemisphere.

Studies of microbial biodiversity are a fundamental starting point for further research into ecosystem function. This project has provided critical baseline information on the composition and distribution of microbial communities in a variety of cave microhabitats and provides a focus for future studies of ecological function such as the microbial contribution to geochemical cycling and mineral precipitation and deposition in the subsurface biosphere. This study has taken significant steps in identifying the microorganisms present in cave environments, and this research now needs to be taken to the next level, ie. what are these bacteria doing and what role do they play in the ecosystem? Cross-disciplinary studies are needed which correlate the presence and distribution of microorganisms with a comprehensive analysis of the habitat at the scale in which microbes function to attempt to understand the daunting complexities of the interactions between microbes and minerals.

158 References

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186 Appendix 1: Media Preparation and Recipes

APPENDICES

Appendix 1: Media Preparation and Recipes

Media Preparation

Culture media was prepared as per manufacturers instructions unless otherwise stated.

Addition of supplements was as per manufacturers instructions or as described in this section.

Where necessary pH was modified by the addition of O.lM NaOH or O.lM HCl as required. All water used in the preparation of media was prepared by glass distillation of tap water.

Sterilisation was by autoclaving at 121°C at 15 psi for 20 min (unless otherwise specified) or in the case of non-sterile, heat sensitive supplements by filter sterilisation. After autoclaving, agar medium was cooled to 55 °C prior to pouring plates. Media were stored at 4 °C for up to 4 wk.

Media containing antibiotics was stored in the dark at 4 °C for up to 2 wk.

Culture Media

Starch-Casein Agar (SC); (Kuster & Williams, 1964).

10 g soluble starch, 0.3 g casein, 2 g KN03, 2 g NaCl, 2 g K2HP04, 0.05 g MgS04.7H20, 0.02 g

CaC03, 0.01gFeS04.7HzO,15gagar,1000 mL distilled water, adjust to pH 7.0-7.2, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin.

Arginine -Vitamin Agar (AV) (Nonomura & Ohara, 1969).

0.3 g L-Arginine, 1 g glucose, 1 g glycerol, 0.3 g K2HP04, 0.2 g MgS04.7HzO, 0.3 g NaCl, 15 g agar, 1000 mL distilled water, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin, 5 mL vitamin solution, 5 mL mineral solution.

Marine Agar (MA)

37.4 g Marine broth (Oxoid 2216), 15 g agar, 1000 mL distilled water, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin.

187 Appendix 1: Media Preparation and Recipes R2A Agar (R2A)

18.1 g R2A agar (Oxoid CM 906), 1000 mL distilled water, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin.

1/2 Strength Tryptone Soya Agar (1/2 TSA)

15 g TSA Broth (Oxoid CM 129), 15 g agar, 1000 mL distilled water, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin.

Oatmeal Agar (OA) (Williams & Wellington, 1982).

20 g commercial blended oats, 800 mL distilled water, autoclave for 30 min at 121 °C, cool to room temperature with occasional vigorous shaking; add 1 g yeast extract, 1 mL mineral solution, 15 g agar, 200 mL distilled water, autoclave for 20 min at 121 °C, cool to -55 °C, add 10 mL Nystatin.

Luria Broth Agar with Ampicillin (LB-AMP)

10 g Bacto®-tryptone, 5 g Bacto®-yeast extract, 5 g NaCl, 15 g agar, 1000 mL distilled water, adjust to pH 7.0, autoclave for 20 min at 121 °C, cool to -55 °C, add ampicillin to a final

1 concentration of 100 µg mL· •

Supplements

Nystatin 0.1 g Nystatin (SIGMA), 20.0 mL Methanol, filter sterilise and store at 4 °C.

AV and OA Mineral Solution

2.0 g Fez(S04).H20, 0.2 g CuS04.5H20, 0.2 g ZnS04.7H20, 0.2 g MnS04.7H20, 200 mL distilled water, filter sterilise and store at 4 °C.

AV Vitamin Solution 0.1 g Thiamine Hydrochloride, 0.1gRiboflavin,0.1gNicotimamide,0.1 g Pyridoxine Hydrochloride, 0.1 g Inositol, 0.1 g Calcium Pantotheenate, 0.1 g Para-Aminobenzoic Acid, 0.05 g Biotin, 200 mL distilled water, filter sterilise and store at 4 °C.

0.1 M iso-propyl-beta-D-thio-galactopyranoside (IPTG)

1.2 g IPTG, 50 mL ddH20, filter-sterilise and store at 4 °C.

5-bromo-4-chloro-3-indoyl-beta-D-thio-galactopyranoside (X-Gal) 100 mg X-Gal, 2 mL N,N' -dimethyl-formamide, cover with foil and store at-20 °C.

188 Appendix 2: Cryopreservation Protocol

Appendix 2: Cryopreservation Protocol

Protocol for Freeze-Drying Bacterial Cultures (pers. comm. Carol Mancuso Nichols, University of Tasmania, 2003)

1) Subculture pure isolate onto solid media and incubate long enough to ensure a good cover of growth.

2) Prepare labels with Strain Identification on one side and date on the other. Make sure ink will not run when wet. Prepare 4-5 ampoules for each strain.

3) Place these labels into glass ampoule (Borosilicate - approx 8mm diam X 105 mm long). Plug ampoule with small piece of cotton wool, rolled for easy removal and replacement.

4) Place ampoules into paper Sterilope for autoclaving and autoclave at 121 °C for 20 min.

5) Prepare 50 ml 1/2 strength seawater (25 ml deionized water+ 25 ml seawater (or equivalent). Autoclave at 121 °C for 20 min. Prepare 50 ml 20% (w:v) solution of skim milk in deionized water and autoclave at 108 °C for 30 min. After cooling combine the solutions.

6) Aseptically, using the skim milk solution, prepare a suspension of cells from agar plate. Add 0.5 ml to each ampoule and replace cotton plug. Try to avoid dripping suspension down the inside of the ampoule, if possible.

7) Place ampoule into circular rack for vacuum centrifugation [Speedivac Centrifigal Freeze Drier, Model 5PS, Edwards High Vacuum, Ltd, Sussex, England], which spins the ampoules that are placed under a glass dome. Turn on vacuum. The centrifugation keeps the liquid milk/ cells from boiling up under vacuum.

8) After 2 hrs, turn off centrifuge and continue drying for a further 6-8 hrs.

9) When the contents of the ampoules is dried, remove ampoules. Using air I gas mix, draw out ampoules in flame so that the section of the ampoule below 2 from the top and above 5-6 cm from the bottom is a narrow capillary.

10) Carefully place ampoule on the 48 port manifold of the freeze-drier. Turn on vacuum. Leave for 1-2 hrs to ensure vacuum.

11) Holding ampoule at the bottom, use flame to seal ampoule at the narrow section while pulling it gently away from the manifold. Ensure the system is still under vacuum before proceeding. Use flame to round any sharp/pointy ends on the ampoule.

12) Open ampoule and add 0.5 ml liquid media. Streak onto agar plate to check for viability and purity.

Bacterial cultures stored at -70°C. Cultures maintained in duplicate, with one set held for subculture purposes only. Vials were placed at -20°C for 24 hrs before transfer to -70°C for storage up to 7 years.

189